Treatment of collagen defects using protein solutions

ABSTRACT

Methods of stimulating collagen production, including stimulation of chondrocyte production, at the site of a defect. Methods include administering to the site of a defect at least two proteins from the group IL-1ra, sTNF-RI, sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII.

INTRODUCTION

The present technology relates to methods of stimulating chondrocytes atthe site of a collagen defect in mammalian subjects. In particular,methods comprise use of solutions comprising cytokines, including suchsolutions derived from blood and other tissues.

Chondrocytes are cells that produce and maintain the cartilaginousmatrix that comprises cartilage. Collagen is the most abundant proteinin mammals, and is present in a variety of tissues, including skin,connective tissue, cartilage, bone, blood vessels and intervertebraldisks. There are a variety of collagen-related disorders, resulting fromdisease as well as trauma to tissues comprising collagen. Diseases mayresult from genetic defects or nutritional deficiencies that affect theproduction of collagen by chondrocytes. Some collagen disorders asassociated with autoimmune diseases, such as rheumatoid arthritis,lupus, and systemic sclerosis, which destroy collagen after it isproduced. Such diseases may be debilitating and painful.

Cartilage is composed of collagen fibers. Thus, collagen disorders mayaffect the composition and function of cartilage in joints and othertissue structures. Cartilage defects can appear as a hole or a tear in acartilage surface and can result from wear, trauma or disease. Sincecartilage has minimal ability to repair itself, even a small cartilagedefect, if left untreated, can hinder the ability of a human or othermammal to move free from pain and can cause deterioration of a jointsurface. Traditional treatments for cartilage defects include trimmingthe defect from the surface of the cartilage using arthroscopic surgery,or repairing the defect with sutures. More recently, treatments forcartilage defects have included the harvesting of cartilage cells, whichare then cultured and implanted back into the cartilage defect toregenerate cartilage.

However, many such treatments may present side effects, and may havelimited long term utility as underlying conditions become worse.Accordingly, there remains a need to develop novel therapies for thetreatment of cartilage and other collagen defects, particularlytherapies that improve efficacy and have reduced side effects.

SUMMARY

The present technology provides methods and therapeutic compositions forthe stimulation of collagen production, including stimulation ofchondrocyte production. Methods include stimulating chondrocyteproduction at a site of a collagen defect in a mammalian subject,comprising administering to the site a blood-derived compositioncomprising at least two proteins selected from the group consisting ofIL-1ra, sTNF-RI, sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF,TGF-β1, and sIL-1RII, wherein the concentration of each protein in thecomposition is greater than the concentration of the protein in normalblood. For example, compositions may comprise

-   -   (a) at least about 10,000 pg/ml IL1-ra;    -   (b) at least about 1,200 pg/ml sTNF-RI; and    -   (c) a protein selected from the group consisting of sTNF-RII,        IGF-I. EGF. HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII,        and mixtures thereof, wherein the protein has a concentration        higher than the protein's baseline concentration in normal        blood.        In some embodiments, the compositions further comprises a        protein selected from the group consisting of sTNF-RII, IGF-I,        EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII, and        mixtures thereof, wherein the concentration of the protein in        the composition is greater than the concentration of the protein        in normal blood. The compositions may comprise        cytokine-producing cells, platelets and combinations thereof.

The present technology also provides methods for making compositions forstimulating chondrocyte product in a mammalian subject, comprising:

-   -   (a) obtaining a cytokine cell suspension from the subject;    -   (b) fractionating the liquid to produce an autologous protein        solution comprising interleukin-1 receptor antagonist;    -   (c) administering the autologous protein solution to the site of        the collagen defect in the subject.        The cytokine cell suspension may comprise whole blood, bone        marrow aspirate, adipose tissue, urine, fractions thereof, and        mixtures thereof. For example, fractionating may comprise        placing blood in a container a separator operable to separate        the blood into two or more fractions; and centrifuging the        separator to create a platelet-rich plasma fraction. The        platelet-rich plasma may be contacted with a solid extraction        material, such as polyacrylamide beads, to form the autologous        protein solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating a method for producing ancytokine composition.

FIG. 2 is a diagram of a fractionation device.

FIG. 3 shows a device for activating a sample to generateanti-inflammatory cytokines, before (FIG. 3A) and after (FIG. 3B)centrifugation.

FIG. 4 is a diagram of a device for generating a blood clot.

FIG. 5 is a diagram of a single device capable of generating a cytokinecomposition.

Corresponding reference numerals indicate corresponding parts throughoutthe several views of the drawings. It should be noted that the figuresset forth herein are intended to exemplify the general characteristicsof materials, compositions, devices, and methods among those of thepresent technology, for the purpose of the description of certainembodiments. These figures may not precisely reflect the characteristicsof any given embodiment, and are not necessarily intended to fullydefine or limit specific embodiments within the scope of thistechnology.

DETAILED DESCRIPTION

The following description of technology is merely exemplary in nature ofthe composition, manufacture and use of one or more inventions, and isnot intended to limit the scope, application, or uses of any specificinvention claimed in this application or in such other applications asmay be filed claiming priority to this application, or patents issuingtherefrom. A non-limiting discussion of terms and phrases intended toaid understanding of the present technology is provided at the end ofthis Detailed Description.

The present technology relates to methods for stimulating collagenproduction, such as by stimulating chondrocyte production, usingcompositions comprising cytokines, for example, interleukin-1 receptorantagonist protein (IL1-ra) and tumor necrosis factor-alfa. In variousembodiments, methods for stimulating collagen defect in a human or othermammalian subject, comprise:

-   -   (a) obtaining a cytokine cell suspension from one or more        mammalian subjects;    -   (b) fractionating the cytokine cell suspension to produce        protein solution comprising one or more proteins, such as        interleukin-1 receptor antagonist; and    -   (c) administering the autologous protein solution to the site of        collage defect in the subject.

Protein Compositions

The present technology provides methods for stimulating chondrocyteproduction in humans or other mammalian subjects using compositions(herein referred to as “Protein Solutions”) comprising proteinsdissolved, suspended or otherwise carried for delivery to a mammaliansubject in a physiologically-acceptable medium. In various embodiments,such compositions comprise proteins (e.g., cytokines) that are native towhole blood in normal mammal subjects. Such compositions may alsocontain viable cells, including platelets, cytokine-producing cells, andcombinations thereof.

In various embodiments, the Protein Solution comprises at least twoproteins selected from the group consisting of IL-1ra (interleukin-1receptor antagonist), sTNF-RI, sTNF-RII (soluble tumor necrosisfactor-receptor 2), IGF-I (insulin-like growth factor 1). EGF (epidermalgrowth factor), HGF (hepatocyte growth factor), PDGF-AB(platelet-derived growth factor AB), PDGF-BB (platelet-derived growthfactor BB), VEGF (vascular endothelial growth factor), TGF-β1(transforming growth factor-β1, and sIL-1RII (soluble interleukin 1receptor II), wherein the concentration of each protein in thecomposition is greater than the concentration of the protein in normalblood. While the concentration of every such protein in the compositionmay be greater than its respective concentrations in in normal blood, itis not necessary that the concentration of more than two of the proteinsbe greater than their respective concentrations in normal blood.

In various embodiments, a Protein Solution comprises the followingcomponents.

TABLE 1 Protein Solution Exemplary Protein Components Normal Whole BloodComponent Composition Concentration Concentration plasma proteins about80 mg/ml or greater about 67 mg/ml (total) about 100 mg/ml or greaterabout 200 mg/ml or greater about 250 mg/ml or greater albumin about 60mg/ml or greater about 56 mg/ml about 100 mg/ml of greater fibrinogenabout 3.2 mg/ml or greater about 2.9 mg/ml about 4 mg/ml or greaterIL-1ra about 10,000 pg/ml or greater about 4200 pg/ml about 25,000 pg/mlor greater about 30,000 pg/ml or greater from about 25,000 to about110,000 pg/ml from about 25,000 to about 40,000 pg/ml sTNF-RI about1,200 pg/ml or greater about 630 pg/ml about 1,800 pg/ml or greaterabout 3,000 pg/ml or greater sTNF-RII about 3,000 pg/ml or greater about1200 pg/ml about 5,000 pg/ml or greater about 7,000 pg/ml or greaterabout 9,000 pg/ml or greater sIL-1RII about 15,000 pg/ml or greaterabout 11,800 pg/ml about 20,000 pg/ml or greater about 25,000 pg/ml orgreater Growth factors EGF about 800 pg/ml or greater about 250 pg/mlabout 1,000 pg/ml or greater about 1,200 pg/ml or greater HGF about1,000 pg/ml or greater about 500 pg/ml about 2,500 pg/ml or greaterabout 2,800 pg/ml or greater about 3,000 pg/ml or greater PDGF-AB about35,000 pg/ml or greater about 6,000 pg/ml about 50,000 pg/ml or greaterabout 70,000 pg/ml or greater PDGF-BB about 10,000 pg/ml or greaterabout 1,500 pg/ml about 15,000 pg/ml or greater about 20,000 pg/ml orgreater TGF-β1 about 100,000 pg/ml or greater about 10,000 pg/ml about150,000 pg/ml or greater about 190,000 pg/ml or greater IGF-1 about130,000 pg/ml or greater about 70,000 pg/ml about 150,000 pg/ml orgreater about 160,000 pg/ml or greater VEGF about 500 pg/ml or greaterabout 150 pg/ml about 600 pg/ml or greater about 800 pg/ml or greaterProtein concentrations can be measured using the methods set forth inExample 4.

The composition further preferably comprises viable cytokine-producingcells, lysed cytokine-producing cells, or both. In a preferredcomposition, the Protein Solution comprises monocytes, granulocytes, andplatelets. In various embodiments, a Protein Solution comprises thefollowing components.

TABLE 2 Protein Solution Exemplary Cellular Components Normal WholeBlood Component Composition Concentration Concentration cytokine- atleast about 15 k/μl 6.5 k/μl producing cells at least about 30 k/μl fromabout 30 to about 60 k/μl from about 40 to about 50 k/μl red blood cellsless than about 3 M/μl 4.5 M/μl less than about 2 M/μl less than about2.5 M/μl platelets at least about 400 k/μl 240 k/μl at least about 800k/μl at least about 1,000 k/μl neutrophils at least about 5 k/μl 3.7k/μl at least about 10 k/μl at least about 12 k/μl monocytes at leastabout 1 k/μl 0.5 k/μl at least about 2 k/μl at least about 3 k/μllymphocytes at least about 5 k/μl 2 k/μl at least about 10 k/μl at leastabout 20 k/μl eosinophiles at least about 0.15 k/μl 0.1 k/μl at leastabout 0.18 k/μl basophils at least about 0.2 k/μl 0.1 k/μl at leastabout 0.4 k/μl at least about 0.6 k/μl

It will be understood that this concentration is species specific.Further, it is understood that concentrations may vary among individualsubjects. Thus, in methods comprising production of a Protein Solutionfrom the blood or other tissue containing cytokine-producing cells, theconcentration of proteins and cells in the Protein Solution may varyfrom those recited above; the values recited above are mean values forconcentrations as may be seen in a population of subjects.

In various embodiments, the concentration of one or more of the proteinsor other components in the Protein Solution is greater than theconcentration of the component in normal blood. (Compositions with suchhigher concentrations of components are said to be “rich” in suchcomponents.) As referred to herein, the concentration of a component in“normal” blood or other tissue is the concentration found in the generalpopulation of mammalian subjects from which the tissue is obtained,e.g., in normal whole blood. In methods wherein the anti-inflammatorycytokine composition is derived from tissue from a specific subject, the“normal” concentration of a protein or cell may be the concentration inthe blood of that individual before processing is performed to derivethe protein or cell.

Thus, in various embodiments, the concentration of one or morecomponents of the Protein Solution is greater than about 1.5 times,about 2 times, or about 3 times, greater than the concentration of thecomponent in normal blood. For example, components may have greaterconcentrations in the compositions, relative to normal (whole) blood, asfollows:

-   -   IL-1ra, at a concentration that is at least about 2.5, or at        least about 3 or at least about 5, times greater;    -   sTNF-RI, at a concentration that is at least about 2, or at        least about 2.5 or at least about 3, times greater;    -   sTNF-RII, at a concentration that is at least about 2, or at        least about 2.5 or at least about 3, times greater;    -   sIL-1RII, at a concentration that is at least about 1.5, or at        least about 1.8 or at least about 2, times greater;    -   EGF, at a concentration that is at least about 2, or at least        about 3 or at least about 5, times greater;    -   HGF, at a concentration that is at least about 2, or at least        about 3 or at least about 4, times greater;    -   PDGF-AB, at a concentration that is at least about 2, or at        least about 3 or at least about 5, times greater;    -   PDGF-BB, at a concentration that is at least about 2, or at        least about 3 or at least about 5, times greater;    -   TGF-β1, at a concentration that is at least about 3, or at least        about 4 or at least about 6, times greater;    -   IGF-1, at a concentration that is at least about 1.2, or at        least about 1.4 or at least about 1.5, times greater,    -   VEGF, at a concentration that is at least about 2, or at least        about 2.5 or at least about 3, times greater;    -   cytokine-producing cells, at a concentration that is at least        about 2, or at least about 3 or at least about 4, times greater,    -   platelets, at a concentration that is at least about 2, or at        least about 3 or at least 4, times greater,    -   neutrophils, at a concentration that is at least 1.5, or at        least 2 or at least 3, times greater;    -   monocytes, at a concentration that is at least 3, or at least 4        or at least 6, times greater,    -   lymphocytes, at a concentration that is at least 5, or at least        8 or at least 10, times greater; and    -   basophils, at a concentration that is at least 2, or at least 4        or at least 6, times greater        Also, the concentration of erythrocytes in the Protein Solution        is preferably at least half, or at least a third, of the        centration of erythrocytes in normal blood.

For example, a Protein Solution may comprise:

(a) at least about 10,000 pg/ml IL1-ra;

(b) at least about 1,200 pg/ml sTNF-RI; and

(c) a protein selected from the group consisting of sTNF-RII, IGF-I,EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1RII, and mixturesthereof, wherein the protein has a concentration higher than theprotein's baseline concentration in normal blood. In another example, aProtein Solution comprises:

-   -   (a) interleukin-1 receptor antagonist (IL-1ra), at a        concentration at least 3 times greater than the concentration of        IL-1ra in normal blood;    -   (b) soluble tissue necrosis factor-r1 (sTNF-r1), at a        concentration at least 2 times greater than the concentration of        IL-1ra in normal blood;    -   (c) cytokine-producing cells at a concentration at least 2 times        greater than the concentration of cytokine-producing cells in        normal blood; and    -   (d) platelets, at a concentration at least 2 times greater than        the concentration of platelets in normal blood.

In some embodiments, the concentration of IL-1ra in the Protein Solutionis preferably at least 5,000, or at least 10,000, times greater than theconcentration of interleukin-1α in the Protein Solution. The ratio ofIL-1ra:interleukin-1β (IL-1 β) concentrations is preferably at least100. In some embodiments, the concentration of IL-1ra in the ProteinSolution is preferably at least 1500, or at least 8000, times greaterthan the concentration of IL-1 β in the Protein Solution. The ratio ofsIL-1RII:interleukin-1β (IL-1 β) concentrations is preferably greaterthan 1. In some embodiments, the sIL-1RII in the Protein Solution ispreferably at least 2000, or at least 45000, times greater theconcentration of interleukin-1β in the Protein Solution.

Methods of Making Protein Solutions

Protein Solutions may be made by any of a variety of methods, includingadmixture of individual components and processes wherein one or morecomponents are derived from a source material. In various embodiments,the Protein Solution is made by fractionating a cytokine cellsuspension, to produce a protein solution comprising IL1-ra.

Obtaining Protein Solutions by Contacting Cytokine-Producing Cells withan Extraction Material

In various embodiments, Protein Solutions are made by derivation of oneor more components from tissue comprising cytokine-producing cells. Asreferred to herein, a “cytokine producing tissue” is a tissue obtainedfrom a mammalian subject, comprising cells that are capable of producingcytokines. Such cells include cytokine-producing cells, adipose stromalcells, bone marrow stromal cells, and combinations thereof. It isunderstood that cytokine-producing cells include monocytes, lymphocytes,and granulocytes such as neutrophils, eosinophils, and basophils. Whiteblood cell useful in the methods of this technology preferably includemonocytes and neutrophils. Cytokine producing tissues among those usefulherein include blood, adipose tissue, bone marrow, and fractionsthereof, as further discussed below.

Blood useful herein includes whole blood, plasma, platelet-rich plasma,platelet-poor plasma, and blot clots. In a preferred embodiment, methodsof the present technology use platelet-rich plasma (PRP), containingcytokine-producing cells and platelets, comprising the buffy coat layercreated by sedimentation of whole blood. Adipose tissue useful hereinincludes any fat tissue, including white and brown adipose tissue, whichmay be derived from subcutaneous, omental/visceral, mammary, gonadal, orother adipose tissue sites. Bone marrow useful herein includes redmarrow and yellow marrow. In a preferred embodiment, bone marrow is bonemarrow concentrate, obtained from the red marrow of long bones,comprising hematopoietic and mesenchymal stems cells. As discussedabove, blood, adipose, and bone marrow tissue useful herein may be fromeither autologous or allogeneic sources, relative to the subject to betreated according to methods of this technology. Compositions may alsobe made from combinations of allogeneic and autologous tissues.

In some embodiments, methods comprise fractionating a liquid (a“cytokine cell suspension.”) comprising cells capable of producingcytokines, such as IL1-ra and sTNF-R1. As discussed above, such cellsinclude cytokine-producing cells, adipose stromal cells, bone marrowstromal cells, and combinations thereof. In some embodiments, thecytokine cell suspension is a liquid comprising cytokine-producingcells. It should be understood that the cytokine cell suspensioncomprises cells and an extra-cellular liquid, regardless of the relativeproportions of the cells and liquid. In some embodiments, the suspensionmay comprise primarily cells, with liquid being present as only a minorcomponent, essentially wetting the cells. In some embodiments, theliquid may comprise two phases, consisting of a phase primarilyconsisting of liquid and a phase primarily consisting of cells, forminga suspension of cells in the liquid only upon agitation or other mixing.

In some embodiments, fractionating a cytokine cell suspension comprisescontacting the liquid with a solid extraction material. As exemplifiedin FIG. 1, such processes comprise:

-   -   (a) obtaining a cytokine cell suspension, such as a liquid        comprising cytokine-producing cells (steps 105, 115 or 135, or        combinations thereof);    -   (b) contacting the tissue with a solid extraction material (step        140); and    -   (c) isolating a protein-containing liquid from the solid        extraction material (step 150).        Obtaining the suspension 105, 115, 135 can comprise any of a        variety of methods for creating a liquid containing cells among        those known in the art. Such methods include isolation from        tissue and culturing. Obtaining may be performed directly in the        method, whereby a health care practitioner or other individual        performs isolation, processing, culturing or other processes for        creating the suspension, in a procedure that includes the        contacting and isolating steps. In some embodiments, the        processes for creating the suspension are performed        contemporaneously with the contacting and isolating steps, as        part of a point-of-care procedure, as discussed further herein.        Alternatively, obtaining the suspension may be indirect,        involving only the acquisition of the suspension for use in the        contacting and isolating steps, wherein the processing to create        the suspension has previously been performed by another party.

In various embodiments, obtaining comprises isolating a cytokine cellsuspension, comprising cytokine-producing cells or othercytokine-producing cells, from blood, adipose tissue, bone marrowaspirate or other tissue comprising cytokine-producing cells, asexemplified in Steps 110, 120 and 125 of FIG. 1. Methods may compriseobtaining a cytokine cell suspension from two, three or more tissuesources.

Obtaining a Liquid Containing Cytokine-Producing Cells from Blood

In embodiments comprising the use of blood, the blood may be useddirectly in contacting the solid extraction material, as exemplified instep 140 of FIG. 1, or may be processed to provide a blood fraction,such as PRP, in a preferred embodiment. Many devices and methods forcreating blood fractions are known in the art, using such means ascentrifugation and filtering.

In various embodiments, methods of the present technology comprisecreating PRP as the cytokine cell suspension, using centrifugation. Suchmethods generally comprise placing blood in a container a separatoroperable to separate the blood into two or more fractions, andcentrifuging the separator to create a platelet-rich plasma fraction.Such devices may include a tube and a buoy disposed in the tube, whereinthe buoy has a density such that the buoy reaches an equilibriumposition upon centrifugation of the tissue in the tube, the equilibriumposition being between a first fraction and a second fraction comprisingcytokine-producing cells, the second fraction having a concentration ofcytokine-producing cells greater than the concentration ofcytokine-producing cells in the first fraction. Such methods furthercomprise centrifuging the tube so that the buoy defines an interfacebetween the first fraction and the second fraction comprisingcytokine-producing cells. The second fraction is then collected forfurther use in the methods of this technology.

One such device useful herein is described in U.S. Pat. No. 7,992,725,Leach et al., issued Aug. 9, 2011. Such a device is commerciallyavailable as GPS III Platelet Concentrate and Separation System, fromBiomet Biologics, LLC (Warsaw, Ind., USA). The device can be used in aclinical or laboratory environment to isolate fractions from asuspension or multi-component tissue material obtained from a subject,such as blood, bone marrow aspirate, cerebrospinal fluid, adiposetissue, Isolated fractions can include platelets, platelet poor plasma,platelet rich plasma and stromal cells. The isolated fractions can eachhave equilibrium point or positions within the separation container thatare achieved when separation has occurred. For example, a buffy coat(PRP) of whole blood may have an equilibrium position above that of thered blood cells when a sample of whole blood is separated.

The fractionation device 200 is exemplified in FIG. 2. The fractionationdevice 200 comprises a buoy 210 and a container wall 215. When theseparation container 205 is centrifuged, the buoy perimeter 210 a andthe container wall 215 have clearance allowing the buoy 210 to movewithin the separation container 205 and a material to pass between thebuoy perimeter 210 a and the container wall 215. Alternatively, the buoy210 could have an opening, such as a centrally or internally locatedopening or a peripheral channel running the height of the buoy, whichwould allow a material to move through the buoy.

The buoy 210 is carried in the separation container 205 and has a tuneddensity that is configured to reach a selected equilibrium position in asuspension. The buoy can have its density tuned in the range from about1.0 g/cc to about 1.10 g/cc, such as about 1.06 g/cc. The buoy 210,according to various embodiments, can be formed to include the tuneddensity and can be formed of one or more materials to achieve the tuneddensity.

Referring to FIG. 2, a collection area 220 is positioned within thedevice 200 after a separation procedure has occurred. The collectionarea 220, defined relative to the buoy 210, is positioned at anequilibrium position of a separated or isolated middle fraction 225 inthe container. The equilibrium position of a selected fraction can bedefined as its position within the container relative to other fractionsin the container of a separated sample or material. The equilibriumposition can also be defined relative to the axis X of the buoy 210 orthe container 12. The equilibrium position, however, may depend upon theamount of the sample of the amount of a selected fraction within asample. According to the illustration in FIG. 2, the equilibriumposition of the fraction 230 is above or nearer a top 235 of the device200 than the equilibrium position of the fraction 225. Thus, the buoy210 can be tuned, such as including a selected density or specificgravity, to position the collection area 220 relative to an equilibriumposition of any selected fraction.

In some embodiments, the buoy 210 can comprise a collection port 240.The collection port 240 communicates with access port 245 andcommunicates with a collection space 220 above buoy upper surface 250and can be located near the buoy perimeter 210 a. In some embodiments,the collection port 240 is not carried on the buoy, but rather thecollection port is a withdraw device such as a syringe that is insertedthrough an access port or top of the device 200.

According to various embodiments, an isolator 255, is coupled to thebuoy 210. The combination of the isolator and buoy, according to variousembodiments, can also be referred to as a separation assembly member.The isolator 255, for example, provides a means for creating thecollection compartment 220 and comprises one or more spacers 260, 265 toposition the isolator 255 apart from the buoy 210 to create thecollection compartment 220. A withdraw port 270 can be carried on theisolator 255 communicating with the withdraw port 245 and the collectionport 240. The spacer 260, 265 can also serve as a conduit 275 betweenthe collection port 50 and a withdraw or withdraw port 245. The withdrawport 245 serves as a structure for withdrawing the isolated or secondfraction 310 from the collection compartment 220.

After centrifuging the device 200 containing whole blood, the firstfraction or top fraction 230, can be platelet-poor-plasma, the middlefraction 225 can be platelet-rich plasma or platelet concentrate, and abottom fraction 278 can be red blood cells. Therefore, the fractionationmethod further comprises withdrawing a desired fraction from the device200. Various ports 205, 245 and 280 can be provided to allow access toany appropriate compartment of the device 200. The access ports 205,245, 280 can be any means that allow communication from outside theseparation device 200 to the device's interior, such as a Luer lockport, a septum, a valve, or other opening. Additionally, collection venttube 285 allows removal of a fractionated suspension in the collectionarea 220 through opening 290 without the need to remove the fraction,such as plasma, above the isolator 255. Although, without a collectionvent tube 285, the fraction above the isolator could be removed and thecollection area could be vented to the area above the isolator.

A method for using the fractionation device 200 can begin by inputtingwhole blood via an access port 205. The fractionation device 200 isplaced into a centrifuge and spun for a period that is appropriate forfractionating whole blood. An exemplary period can be for about fiveminutes to about twenty minutes at a rate of about 320 rpm to about 5000rpm. This speed may produce a selected gravity that may be approximately7.17×g to about 1750×g (times greater than the normal force of gravity).

Other devices that may be used to isolate platelet-rich plasmadescribed, for example, in U.S. Pat. No. 5,585,007, Antanavich, issuedDec. 17, 1996; U.S. Pat. No. 6,398,972, Blasetti et al., issued Jun. 4,2002; U.S. Pat. No. 6,649,072, Brandt et al., issued Nov. 18, 2003; U.S.Pat. No. 6,790,371, Dolocek, issued Sep. 14, 2004; U.S. Pat. No.7,011,852, Sukavaneshvar et al., issued Mar. 14, 2006; U.S. Pat. No.7,179,391, Leach et al., issued Feb. 20, 2007; U.S. Pat. No. 7,374,678,Leach et al., issued May 20, 2008; U.S. Pat. No. 7,223,346, Dorian etal., issued May 29, 2007; and U.S. Pat. No. 7,708,152, Dorian et al.,issued May 4, 2010.

In addition to the GPS® Platelet Concentrate and Separation Systems, avariety of other commercially available devices may be used to isolateplatelet-rich plasma, including the Magellan™ Autologous PlateletSeparator System, commercially available from Medtronic, Inc.(Minneapolis, Minn., USA); SmartPReP™, commercially available fromHarvest Technologies Corporation (Plymouth, Mass., USA); the AutoloGel™Process, commercially available from Cytomedix, Inc. (Rockville, Md.,USA); the GenesisCS System, commercially available from EmCyteCorporation (Fort Myers, Fla., USA); and the PCCS System, commerciallyavailable from Biomet 3i, Inc. (Palm Beach Gardens, Fla., USA); and theArthrex ACP™ Double Syringe System, commercially available from Arthrex,Inc. (Naples, Fla., USA).

Referring again to FIG. 1, blood drawn from the patient may be mixedwith an anticoagulant in one or more of Steps 115, 120, 125, and 130, soas to facilitate processing. Suitable anticoagulants include heparin,citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid(EDTA), anticoagulant citrate dextrose solution (ACD), and mixturesthereof. For example, the anticoagulant may be placed in the syringeused for drawing blood from the subject, or may be mixed with the bloodafter it is drawn.

A liquid containing cytokine-producing cells may be prepared by admixingcells with a suitable liquid, as shown in step 125, using methods knownin the art. For example, cytokine-producing cells may be isolated fromwhole blood by lysing red blood cells or by centrifugation of wholeblood utilizing a density gradient where the cytokine-producing cellssediment to the bottom of a centrifuge tube. An example of densitycentrifugation includes the Ficoll-Paque™ Plus (GE HealthcareBio-Sciences, Piscataway, N.J., USA). In some cases, a density gradientmay be used to further separate mononuclear and polymorphonuclear cells.Cytokine-producing cells may also be prepared from whole blood usingfiltration; an example includes the Acelere™ MNC Harvest System (PallLife Sciences, Ann Arbor, Mich., USA). Cytokine-producing cells can alsobe obtained from bone marrow. The cytokine-producing cells may be thensuspended in a suitable medium, such as plasma, so as to maintain theirviability.

Other methods may be used to create platelet-rich plasma or other liquidcontaining cytokine-producing cells. For example, whole blood can becentrifuged without using a buoy system, whole blood may be centrifugedin multiple stages, continuous-flow centrifugation can be used, andfiltration can also be used. In addition, a blood component includingplatelet-rich plasma can be produced by separating plasma from red bloodcells using a slow speed centrifugation step to prevent pelleting of theplatelets. In other embodiments, the buffy coat fraction formed fromcentrifuged blood can be separated from remaining plasma andre-suspended to form platelet-rich plasma.

Obtaining a Cytokine Cell Suspension from Adipose Tissue

In embodiments comprising the use of adipose tissue, the adipose tissuemay be used directly in contacting the solid extraction material, asexemplified in step 140 of FIG. 1, or the adipose tissue may beprocessed to provide isolated adipocytes in step 110. Cell fractionscomprising adipose-derived stem cells are also useful in this method. Insome embodiments, adipose tissue is derived from human subcutaneous fatisolated by suction assisted lipectomy or liposuction. Stromal cells maybe isolated from the adipose tissue and/or tissue portions using anysuitable method, including methods known in the art such as mechanicaland breakdown centrifugation. Stromal cells can also be isolated usingenzymatic digestion. For example, stromal cells can be isolated fromlipoaspirate, treated by sonication and/or enzymatic digestion, andenriched by centrifugation. Stromal cells isolated from adipose tissuemay be washed and pelleted.

For example, adipose tissue can be collected by suction-assistedtumescent liposuction inside a specialized collection container attachedto suction hoses and to a liposuction cannula. The collection containercan have a gauze-type grid filter that allows the tumescent fluid topass through and retains the solid adipose tissue. After collecting theadipose tissue, the collection container is removed from the suctiondevice and reattached to a centrifugation device. The filter unit mayfurther contain a filter having approximately a 100 micrometer poresize. Once the collection container containing the adipose tissue isattached to the centrifugation device, the tissue is sonicated. Aftersonication, the entire apparatus is inserted into a centrifuge bucketand centrifuged at, for example, 300×g for 5 minutes. Aftercentrifugation, the collection container together with the filter unitis detached and can be discarded. The pellet containing the stromalcells can then be re-suspended in biocompatible solutions, such asplasma, plasma concentrate and platelet-rich plasma.

Various methods and devices for isolating and/or fractionating adiposetissue and adipocytes include those as described by U.S. Pat. No.7,374,678, Leach, issued May 20, 2008; U.S. Pat. No. 7,179,391 to Leachet al., issued Feb. 20, 2007; U.S. Pat. No. 7,992,725, Leach et al.,issued Aug. 9, 2011; U.S. Pat. No. 7,806,276, Leach et al., issued Oct.5, 2010; and U.S. Pat. No. 8,048,297, Leach et al., issued Nov. 1, 2011.A device, such as the GPS™ Platelet Concentrate System, commerciallyavailable from Biomet Biologics, LLC (Warsaw, Ind., USA), may be used toisolate adipocytes.

Obtaining a Liquid Containing Cytokine-Producing Cells from Bone Marrow

In embodiments comprising the use of bone marrow, the marrow may be useddirectly in contacting the solid extraction material, as exemplified instep 140 of FIG. 1, or may be processed to provide a bone marrowconcentrate, as in step 135. Many devices and methods for obtaining andconcentrating bone marrow are known in the art.

An exemplary process for isolating and creating a bone marrowconcentrate (cBMA) is diagrammed in FIG. 6. Generally, the method 600may start in step 605 with obtaining a bone marrow aspirate volume. Thebone marrow aspirate (BMA) may be obtained in any selected or generallyknown manner. For example, a selected region of bone, such as a portionnear an operative procedure, may be used to obtain the bone marrowaspirate. Generally, an accessing device, such as a syringe and needle,may be used to access an intramedullary area of a selected bone. A smallvolume of the selected portion may be drawn from a plurality oflocations to obtain an appropriate volume of BMA or selected fraction ofthe BMA.

Once a selected volume of the BMA is obtained in step 605, it may beseparated and concentrated using a gravimetric separator. Separatorsamong those useful herein are operable to separate a multi-componentfluid that generally includes various components or constituents ofvarying densities that are commingled or mixed together, including thosedescribed above for separation of fractions from blood and adiposetissue. The separator may include a buoy that is of a selected densityrelative to BMA. Such separators include those described above for usein concentrating and isolating fractions from blood and adipose tissue,including those described in U.S. Pat. No. 7,374,678. Leach, issued May20, 2008; U.S. Pat. No. 7,179,391 to Leach et al., issued Feb. 20, 2007;U.S. Pat. No. 7,992,725, Leach et al., issued Aug. 9, 2011; U.S. Pat.No. 7,806,276, Leach et al., issued Oct. 5, 2010; and U.S. Pat. No.8,048,297, Leach et al., issued Nov. 1, 2011. A device, such as the GPS™Platelet Concentrate System, commercially available from BiometBiologics, LLC (Warsaw, Ind., USA), may be used to isolate adipocytes.Separators and methods that may be used to fractionate BMA at steps 610and 615 are also described, for example, in U.S. Application PublicationNo. 2006/0278588, Woodell-May, published Dec. 14, 2006. The BMA may bepositioned in a separator according to various embodiments in step 610.Once the BMA is positioned in the separator, a selected fraction of theBMA may be separated from the BMA in step 615.

Once the BMA is placed in the separator, separator is spun in acentrifuge in a range between about 1,000 and about 8,000 RPM. Thisproduces a force between about 65 and about 4500 times greater than theforce of normal gravity, as generally calculated in the art, on theseparator and the BMA. At this force, the more dense material in a BMAsample is forced toward the bottom end of the tube. The separator canthus be used to remove nucleated cells from the bone marrow sample. Invarious embodiments, concentrated BMA has a concentration of nucleatedcells that is at least 2, at least 3, at least 4, or at least 5 timesthe concentration of nucleated cells in BMA.

Obtaining a Liquid Containing Cytokine-Producing Cells from Blood Clots

In other embodiments comprising the use of blood, a liquid comprisingcytokine-producing cells may trapped in a blood clot. Cell releasate canbe generated from the blood clot by either compression (“squeezing”),clot disruption, or centrifugation. The blood clot can be made with orwithout anticoagulant and with or without exogenous thrombin bycombining blood or a blood fraction with a clotting agent. Suitableclotting agents include thrombin (e.g., bovine, recombinant human,pooled human, or autologous), autologous clotting protein, andpolyethylene glycol. Calcium may be in the form of a calcium salt, suchas calcium chloride.

In some embodiments, the clotting agent comprises a clotting protein,which may be a clotting fraction derived from a blood obtained from thepatient to be treated. A suitable clotting fraction can be obtained by aprocess of: loading whole blood or plasma with a calcium solution (e.g.,calcium chloride in ethanol) into a blood isolation device; optionallyheating the whole blood or plasma for at least about 20 minutes, at atemperature of at least about 20° C.; and isolating the clottingfraction. The isolating may be performed by centrifuging the heatedwhole blood or plasma. A suitable isolation device is commerciallyavailable as the Clotalyst™ Autologous Thrombin Collection System(hereinafter “Clotalyst System”), sold by Biomet Biologics LLC, Warsaw,Ind., USA.

An exemplary procedure for producing a clotting agent using a device 400of FIG. 4 begins with injecting a reagent comprising calcium chlorideand ethanol into the main chamber 405 through the first port 410. Glassbeads are also placed in the main chamber 405. After the reagent hasbeen injected, the first port 410 is closed using the first replacementcap 415. Blood with anticoagulant is injected into the main chamber 405through the second port 420. After the blood has been injected, thesecond port 420 is closed using the second replacement cap 425.Optionally, the syringes and blood separation device 400 are pre-heatedto a temperature of about 25° C.

The contents of the blood component separation device 400 are mixed byrepeatedly inverting the device 400. e.g. about twelve times, so as tocontact the blood with the glass beads. After mixing, the device isincubated The incubation process can be at a temperature and for aduration that will permit the contents of the device 400 to be heated atabout 25° C. for about 15 minutes. Upon completion of the incubationperiod, a clotted mass of red blood cells, blood plasma, and glass beadsforms at a second end 406 of the main chamber 405. After incubation iscomplete, the device 400 is shaken enough to dislodge and break-up anygel that may be present.

Obtaining a Liquid Containing Cytokine-Producing Cells UsingNon-Centrifugal Methods

As noted above, the liquid containing cytokine-producing cells can beobtained by non-centrifugal means, such as by culturing. As referred toherein, a “non-centrifugal method” comprises a process for obtainingtissue fractions comprising cytokine-producing cells from tissue withoutuse of a centrifuge. In some embodiments, methods are “non-gravimetric,”wherein, based on physical, chemical or physicochemical properties ofthe cells other than density, wherein the concentration of white bloodcells in the fraction are higher than the concentration of white bloodcells in the tissue. Such non-gravimetric methods are, in particular,distinguished from methods wherein a white blood cell fraction iscreated by centrifugation of whole blood or other tissue. In someembodiments, the non-centrifugal method comprises a process solely basedon such properties of white blood cells other than density.Non-centrifugal methods include filtration, antibody binding, andelectrophoretic methods.

For example, as discussed above, cytokine-producing cells may beprepared from whole blood, bone marrow aspirate or other tissue, usingfiltration. Cytokine-producing cells and other cytokine-producing cellsobtained from blood, bone marrow, adipose tissue or other sources mayalso be cultured, using methods among those known in the art. The cellsmay be then suspended in a suitable medium, such as plasma, so as tomaintain their viability and facilitate mixing or other contact with asolid extraction material. A liquid containing the cells may also beproduced by compression or disruption of blood clots, as describedabove.

Contacting a Liquid Cytokine Cell Suspension with an Extraction Materialand Isolating a Protein Solution

In further reference to the exemplified process of FIG. 1, the cytokinecell suspension is incubated or otherwise contacted with a solidextraction material (step 140) to produce a protein-containing liquid.This liquid is then isolated (step 150) from the solid extractionmaterial, as a Protein Solution of the present technology. Withoutlimiting the scope, mechanism or function of the present technology,solid extraction materials useful herein concentrate cytokines or otherproteins in the liquid volume of cytokine-producing cells and may, insome embodiments, activate, stimulate or otherwise increase productionof cytokines, including IL-1ra. Thus, in some embodiments, methodscomprising activating a cytokine cell suspension with a solid extractionmaterial.

The solid extraction material can include various materials that providea particular surface area to contact the cells. The solid extractionmaterial may be a continuous material or may be discontinuous andcomprise a plurality of separate particles. For example, the solidextraction material may be in the form of a plurality of beads, fibers,powder, a porous material, or a surface of a container comprising theliquid containing the cells. The solid extraction material may comprisegeometric forms having various cross-sectional shapes, such asspherical, oval, or polygonal, among others. The solid extractionmaterial can also comprise a continuous porous network, similar to asponge, or can include a plurality of individual porous particles. Thesolid extraction material may also provide a larger surface area bybeing porous in comparison to a non-porous material.

In some embodiments, the solid extraction material includes particleshaving a large aspect ratio, for example, where the particles areneedle-like in shape. The solid extraction material may also be formedas long fibers and may be or take a form similar to glass wool.

In some cases, the solid extraction material can comprise the internalwalls of a container holding the cytokine cell suspension. For example,the solid extraction material may comprise the lumen of a syringe thatcontains the cytokine cell suspension. Other containers include tubes,such as centrifuge tubes, or a blood fractionation device orconcentrator assembly as described elsewhere herein.

Where the solid extraction material is a continuous material, such as aporous sponge-like material, the solid extraction material can be usedin an amount sufficient to absorb or adsorb or include substantially theentire liquid volume of cytokine-producing cells within the pores orinterstices of the solid extraction material. Where the solid extractionmaterial is a discontinuous material, such as a plurality of particles,the solid extraction material can be combined with the liquid containingthe cells to form a slurry-like composition. The slurry can vary inconsistency from paste-like, having a high-solids fraction, to a readilyflowable slurry having a low-solids fraction.

The solid extraction material can provide a large surface area withwhich to contact the cells. However, in some cases, the solid extractionmaterial can be further treated to increase its surface area, forexample, by physically or chemically etching or eroding the surface ofthe solid extraction material. With respect to chemical etching, acorrosive agent can be used to modify the surface of the solidextraction material depending on the nature of the material. Themodified surface may be produced by employing an alkali or an acid, forexample chromosulphonic acid, in particular about 20% to about 80% instrength, preferably about 50% chromosulphonic acid. The solidextraction material can be incubated with the corrosive agent for about5 min to about 30 min in order to chemically etch the surface andincrease the surface area. The solid extraction material can then bewashed to remove the corrosive agent. For example, the solid extractionmaterial can include the internal walls of a container for holding thecytokine cell suspension where the internal walls are etched tosubsequently increase the surface area in contact with the liquid.

Various polymers, metals, ceramics, and glasses can be used as the solidextraction material. In some embodiments, the solid extraction materialcomprises a hygroscopic material. Examples of suitable solid extractionmaterial materials include glasses, minerals, polymers, metals, andpolysaccharides. Minerals include corundum and quartz. Polymers includepolystyrene, polyethylene, polyvinyl chloride, polypropylene, andpolyacrylamide. Metals include titanium. Polysaccharides include dextranand agarose. A preferred solid extraction material comprises, orconsists essentially of, polyacrylamide, as further described below.

The solid extraction material may comprise, for example, continuoussolid extraction material of glass or a plurality of glass particles,glass wool, a continuous solid extraction material of metal such astitanium, a plurality of metal beads, metal powder, and combinationsthereof. A continuous solid extraction material of metal can include ablock or other three-dimensional shape formed of porous metal or metalalloys with an open cell structure. The solid extraction material mayinclude various beads or particles of various sizes includingsubstantially spherical beads. Beads include polystyrene beads,polyacrylamide beads, glass beads, metal (e.g., titanium) beads, or anyother appropriate beads. Beads may be any size appropriate for thecontainer and the amount of cytokine cell suspension being used. In someinstances, bead sizes can range from about 0.001 millimeters to about 3millimeters in diameter. Where the bead size is sufficiently small, thebeads can appear more like a powder.

Polyacrylamide beads used as the solid extraction material can be formedby polymerizing acrylamide monomer using controlled and standardizedprotocols as known in the art to produce relatively uniform beads formedof polyacrylamide gel. In general, polyacrylamide is formed bypolymerizing acrylamide with a suitable bifunctional crosslinking agent,most commonly N,N′-methylenebisacrylamide (bisacrylamide). Gelpolymerization is usually initiated with ammonium persulfate and thereaction rate is accelerated by the addition of a catalyst, such asN,N,N′,N′-tetramethylethylenediamine (TEMED). In various embodiments,polyacrylamide beads comprise 0.5 micromole of carboxyl groups permilliliter of beads, imparting a slight anionic character (negativecharge). The beads are also typically resistant to changes in pH, andare stable in many aqueous and organic solutions. By adjusting the totalacrylamide concentration, the polyacrylamide gel can be formed in a widerange of pore sizes. Moreover, the polyacrylamide beads can be formed inmany sizes and can have relatively uniform size distributions. Bead sizemay range from several micrometers in diameter to several millimeters indiameter. For example, various types of Bio-Gel™ P polyacrylamide gelbeads (Bio-Rad Laboratories, Hercules, Calif., USA) have particle sizesranging from less than about 45 μm up to about 180 μm. Polyacrylamidebeads are also available from SNF Floerger (Riceboro, Ga., USA), PierceBiotechnology, Inc. (Rockford, Ill., USA), and Polymers, Inc.(Fayetteville, Ark., USA).

Once polymerized, polyacrylamide beads can be dried and stored in apowder-like form. The dry beads are insoluble in water but can swellconsiderably upon being rehydrated. Rehydration returns thepolyacrylamide beads to a gel consistency that can be from about two toabout three times the dry state size. Thus, dry polyacrylamide beads(i.e., desiccating polyacrylamide beads) may be used to absorb a portionof a liquid volume, including solutes smaller than the bead pore size,and can serve to concentrate IL-1ra and other proteins produced by thecytokine-producing cells. For example, combining dry polyacrylamidebeads with the blood and/or platelet-rich plasma in step 230 activatesproduction of IL-1ra by the cytokine-producing cells and also reducesthe total liquid volume as the dry beads rehydrate and swell.

Without limiting the scope, mechanism or function of the presenttechnology, it has been discovered that surface contact with the solidextraction material can activate the cells and the solid extractionmaterial can, in some cases, assist in the separation and concentrationof the resulting Protein Solution rich in cytokines, including IL-1ra.For example, in the case of a porous solid extraction material, aportion of the liquid comprising the cells can enter the pores andremain therein. Cells in the liquid may contact this additional surfacearea. In some embodiments, the pores are too small for the cells toenter, but a portion of the liquid can enter the pores. Liquid can beremoved from the solid extraction material and pores by centrifuging,for example.

The solid extraction material is preferably sterilized, using techniquesamong known in the art, in order to prevent contamination of thecytokine cell suspension. For example, heat and pressure sterilizationmethods, such as autoclaving, may be used depending on the particularcomposition of the solid extraction material. Alternative methods, suchas chemical sterilization or irradiation, can be used where the solidextraction material may be adversely affected by the autoclavingprocess.

In some embodiments, the cytokine cell suspension is incubated withsolid extraction material for a time effective to remove a portion ofthe liquid. The incubation may be carried out over a period from about30 seconds to about 72 hours and may be carried out at a temperaturefrom about 20° C. to about 41° C. For example, the incubation may be 24hours or less, 10 hours or less, 5 hours or less, 2 hours or less, 1hour or less, 30 minutes or less, 15 minutes or less 10 minutes or less,5 minutes or less, 4 minutes or less, 3, minutes or less, or 2 minutesor less. Incubation may be at least about 15 seconds, at least about 30seconds, at least about 1 minutes, at least about 90 seconds, at leastabout 2 minutes, at least about 10 minutes, or at least about 30minutes. In some embodiments, incubation s from about 1 minute to about3 minutes. In some embodiments the liquid is not incubated, but iscontacted with the solid extraction material for only so long asnecessary to perform subsequent processing. The contacting may occur atambient conditions, e.g., at a temperature of about 20-25° C.

In some embodiments, the cytokine cell suspension and the solidextraction material are agitated to more thoroughly mix these componentsduring contact. The agitation may be accomplished by inverting, shaking,rocking, stirring, or vortexing the liquid and solid extractionmaterial. Agitation may increase contact of the cells within the liquidwith the solid extraction material. Agitation may be performed once,repeated multiple times, repeated periodically, or may be continuous.The liquid comprising the cells and the solid extraction material mayalso be agitated while the liquid is stimulated with the electromagneticfield. Additional aspects and features relating to producingprotein-rich solutions using polyacrylamide beads and other solidextraction materials are described in: U.S. Patent ApplicationPublication No. 2009/0220482, Higgins et al., published Sep. 3, 2009;U.S. Patent Application Publication No. 2010/0055087, Higgins et al.,published Mar. 4, 2010; U.S. Patent Application Publication2011/0052561, Hoeppner, published Mar. 3, 2011; InternationalApplication Publication 2012/030593, Higgins et al., published Mar. 8,2012; and U.S. Patent Application Publication 2012/0172836, Higgins etal., published Jul. 5, 2012. Compositions and methods useful in aspectsof the present technology are also described in the followingapplications filed concurrently with this disclosure: U.S. patentapplication Ser. No. ______ (Attorney Docket 5490-000959), Binder et al.Methods and Non-Immunugenic Compositions for Treating InflammatoryDiseases; U.S. patent application Ser. No. ______ (Attorney Docket5490-000973), Landrigan, et al., Treatment of Inflammatory RespiratoryDisease Using Protein Solutions; U.S. patent application Ser. No.______(Attorney Docket 5490-001016), Woodell-May et al., Methods andAcellular Compositions for Treating Inflammatory Disorders; U.S. patentapplication Ser. No. ______, O'Shaughnessey, et al. (Attorney Docket5490-001017), Treatment of Pain Using Protein Solutions; U.S. patentapplication Ser. No. ______ (Attorney Docket 5490-001018), Leach et al.,Methods for Making Cytokine Compositions from Tissue UsingNon-Centrifugal Methods; and U.S. patent application Ser. No. ______(Attorney Docket 5490-001023), Landrigan, et al., Treatment ofPeripheral Vascular Disease Using Protein Solutions, all of which areincorporated by reference herein.

Contacting of the liquid containing cytokine-producing cells with thesolid extraction material may be performed using a suitable container orother apparatus to effect the contact. Contacting may be performed in acontinuous process wherein a flow of the liquid is passed over orthrough the solid extraction material, or the liquid and solidextraction material may be contained in a vessel. As discussed above,the vessel may comprise the solid extraction material, or may merelyserve as a container holding the beads or other forms of the material.Containers useful in the present technology include those known in theart, such as the Plasmax™ Plus Plasma Concentrator, commerciallyavailable from Biomet Biologics, LLC (Warsaw, Ind., USA) and may includethose devices and methods of use as described in U.S. Pat. No.7,553,413, Dorian et al., issued Jun. 30, 2009; and U.S. Pat. No.7,694,828, Swift et al., issued Apr. 13, 2010.

Such a device is shown in FIGS. 3A and 3B, for exemplary use with apolyacrylamide gel bead solid extraction material. The device 300 has anupper chamber 305 and a lower chamber 310. The upper chamber 305 has anend wall 315 through which the agitator stem 320 of a gel bead agitator325 extends. The device 300 also has an inlet port 330 that extendsthrough the end wall 315 and into the upper chamber 305. The device 300also includes an outlet port 335 that communicates with a plasmaconcentrate conduit 340. The floor of upper chamber 305 includes afilter 345, the upper surface of which supports desiccated concentratingpolyacrylamide beads 350.

During use, a fluid 355 containing cytokine-producing cells and,optionally, platelets is injected to the upper chamber 305 via the inletport 330 and mixed with the polyacrylamide beads 350. The fluid 355 andpolyacrylamide beads 350 may be mixed by rotating the agitator stem 320and the gel bead agitator 325, to help mix the fluid 355 and beads 350.The mixed fluid 355 and polyacrylamide beads 350 are then incubated forthe desired time at the desired temperature. The device 300 is thencentrifuged so that liquid passes to the lower chamber 310 while thepolyacrylamide beads 350 are retained by a filter 345, therebyseparating the polyacrylamide beads 350 from the resulting solution 360of IL-1ra and other proteins that collects in the lower chamber 310. Thesolution 360 may be removed from the device via outlet port 335.

In some embodiments, a Protein Solution can be made in a process whereina liquid containing cytokine-producing cells is isolated from a tissueand then contacted with a solid extraction material in a continuousprocess. Referring again to FIG. 1, in some embodiments the isolating110, 120, 135 and contacting 140 are performed using a single apparatus,referred to herein as a single separation and concentration device (“S/Cdevice”). One such device is described in U.S. patent application Ser.No. 13/434,245, O'Connell, filed Mar. 29, 2012.

The S/C device comprises a separation region, a first concentrationregion, a second concentration region, a buoy system, an inlet port, acheck valve, a first withdrawal port and a second withdrawal port. FIG.5 shows an S/C device 500 capable of generating an anti-inflammatorycytokine composition from whole blood. For example, the method may startwith obtaining a volume of whole blood, which is filled into aseparation region 505 of the S/C device 500 by injecting through theinlet port 510. A buoy system 515 is located within the separationregion 505. The buoy system comprises a first buoy member 520, a secondbuoy member 525, and a third buoy member 530 that couples the first buoymember 520 to the second buoy member 525. A space between the first andsecond buoy members 520, 525 defines a buoy separation region 535. Adensity of each buoy member can be selected depending on what bloodfraction is desired as a result of a separation. The buoy system 515 caninclude a selected buoy system, such as the buoy system generally usedin the GPS® II or GPS®III gravity platelet separation system sold byBiomet Biologics, LLC. (Warsaw, Ind., USA). Buoy systems are disclosedin U.S. Pat. No. 7,845,499 and U.S. Pat. No. 7,806,276, and U.S. Pat.No. 7,992,725.

A method for obtaining a Protein Solution comprises spinning the S/Cdevice 500 by centrifugation. Centrifugal forces allow the buoy system515 to move through the whole blood, resulting in a fraction of thewhole blood to be located in the buoy separation region 535. Forexample, this fraction may comprise platelet-rich plasma. With a use ofa withdrawal syringe, the selected fraction can be removed from thecollection volume 535 through the third buoy member 530 that defines aremoval passage 540 that is connected with collection face passages 545.A connection elbow 550 can interconnect with the removal passage 540 toallow a vacuum to be formed through the connection elbow 550, thecollection passage 540, and the buoy collection passages 545. Acollection tube 555 can interconnect the connection elbow 550 with awithdrawal elbow 560 that extends from a wall 565 that can be a bottomwall of concentration region 570. A second withdrawal tube 575 can befirst connected with a check valve assembly 580 and a first withdrawalport 585. The first withdrawal port 585 can be connected with thewithdrawal syringe with a Luer lock type connection or other appropriateconnection.

The check valve assembly 580 ensures the fraction being removed flows inone direction and prevents the fraction being removed from reenteringthe second withdrawal tube 575. Furthermore, when material is pushedback into the check valve assembly 580 from the first withdrawal port585, such that material will enter the concentration region 570, a discwithin the check valve 580 can flex down towards the second withdrawaltube 575 and close an opening and thereby open a second opening withinthe check valve assembly 580. The second opening allows the fraction tobe pushed into the concentration region 570.

Therefore, the blood fraction is then re-injected through the firstwithdrawal port 285, through the check valve assembly 580, and into anupper volume 588 of the concentration region 570. Polyacrylamide beads590 are added to the blood fraction in the upper volume 588 and theblood fraction and the polyacrylamide beads 590 can be mixed by shaking.Optionally, the blood fraction and the beads 590 can be incubated for aselected period of time before proceeding with the method.

The method comprises a second step of spinning by centrifugation. Duringthe second centrifugation, the anti-inflammatory cytokine composition isseparated from the beads 590 by being forced through a filter 592 andinto a lower concentration region 595 of the concentration region 570.The Protein Solution can be withdrawn through a third withdrawal tube596 and out a second withdrawal port 598 by use of a second withdrawalsyringe. Again, the syringe can be connected to the second withdrawalport by a Luer® lock type connection.

Referring again to FIG. 1, following contacting the liquid with thesolid extraction materials, a Protein Solution is isolated, as indicatedat step 150. Isolation may be accomplished by drawing off at least aportion of the liquid volume and leaving the beads. In some cases, theextraction material may be sedimented by centrifugation prior to drawingoff the Protein Solution. Isolation may also be performed by filtration,where the material is retained by a filter and the Protein Solutionpasses through the filter using centrifugal force or by using vacuum,for example. If the incubation with extraction material utilizes drypolyacrylamide beads, the liquid volume may be reduced as the beadsswell upon rehydration, thereby concentrating the resulting ProteinSolution. To maintain the increased concentration, care should be takenin the isolation step so as to avoid compressing the beads or drawingliquid out from the swollen beads. For example, high centrifugal forceor high vacuum may collapse the beads and/or draw liquid out of theinternal volume of the beads.

Optional Electromagnetic Stimulation

The cytokine cell suspension can be stimulated with an electromagneticfield, before or during the contacting of the liquid with a solidextraction material. Thus, in some embodiments, stimulation of theliquid comprising the cells can be performed prior to contacting theliquid and the solid extraction material. However, it is preferred thatat least a portion of the contacting step and at least a portion of thestimulating step overlap in time such that the liquid comprising thecells is concurrently in contact with the solid extraction material andstimulated with the electromagnetic field.

Stimulating the cytokine cell suspension with an electromagnetic fieldmay involve various forms of electromagnetic stimulation, such as apulsed electromagnetic field or a capacitively coupled electromagneticfield. In some embodiments, the liquid is stimulated using a powersource coupled to a stimulation coil. The current passing through thecoil produces a pulsing magnetic field which induces in the liquid apulsing electric field. The coil may partially surround the liquid as itis held within a container, such as a tube or syringe. The coil may beintegrated into to the container holding the cytokine cell suspension ormay be removable. For example, a plastic tube can be formed with anintegrated coil or the coil can be temporarily coupled to the containeror placed within the container, for example, the tube can be configuredso that the coil can be snapped onto the container. The power source canbe coupled to the coil as needed to perform the stimulating step.

Stimulation of the liquid with an electromagnetic field may also includeplacing at least two electrodes across the liquid. Electrical energy maythen be applied to the electrodes so as to capacitively couple theelectrodes and generate the electromagnetic field there between. Theelectromagnetic field is therefore able to pass through the liquid so asto increase the rate and/or amount of cytokine production. In otherembodiments, electrodes can be used to produce a direct current or oneor more coils can be used to produce a pulsed electromagnetic field.

The strength of the electromagnetic field during stimulation can be atleast about 0.5 microvolts per centimeter, whether produced by directcurrent, capacitively coupled current, or pulsed electromagnetic field.In the case of a direct current electrode, the amplitude of the currentcan be from about 1 to about 200 microamperes, and in some embodiments,the amplitude may be from about 20 to about 100 microamperes. In stillfurther embodiments, the current may be about 20, about 60, or about 100microamperes. It should be understood, however, that the amplitude ofthe current may be of other suitable magnitudes.

The electromagnetic field applied during the stimulating step may beconstant or vary over time. For example, a sinusoidal time varyingelectromagnetic field can be applied using the electrodes placed acrossthe liquid. Such a sinusoidal time varying electromagnetic field canhave a peak voltage across the electrodes from about 1 volt to about 10volts, and in some embodiments, the peak voltage can be about 5 volts.The corresponding electric field produced can have an amplitude of fromabout 0.1 millivolt per centimeter (mV/cm) to about 100 mV/cm, and insome embodiments can be about 20 mV/cm. The sinusoidal time varyingelectric field may have a frequency of from about 1,000 Hz to about200,000 Hz, and in some embodiments the frequency may be about 60,000Hz.

The electromagnetic field applied to the liquid may also be a pulsedelectromagnetic field. The pulsed electromagnetic field can be inducedusing an external coil and a pulse generator. In this regard, a pulsedelectromagnetic field may have a pulse duration of from about 10microseconds per pulse to about 2000 microseconds per pulse. The pulseduration in one embodiment can be about 225 microseconds. The pulses mayinclude electromagnetic bursts, in which a burst can comprise from 1pulse to about 200 pulses. Alternatively, the electromagnetic field mayhave bursts that comprise from about 10 pulses to about 30 pulses. Inthis regard, in one embodiment each burst may comprise about 20 pulses.

The frequency at which bursts in the pulsed electromagnetic are appliedmay vary. In this regard, bursts can be repeated at a frequency of fromabout 1 Hz to about 100 Hz in some embodiments, and can be repeated at afrequency of about 10 Hz to about 20 Hz in other embodiments.Furthermore, bursts can repeat at a frequency of about 1.5 Hz, about 15Hz or about 76 Hz. A burst can have a duration from about 10microseconds up to about 40.000 microseconds. In this regard, a burstcan have a duration of about 4.5 milliseconds.

Suitable devices for generating a capacitively coupled electromagneticfield include SpinalPak® spinal stimulator (EBI, L.P., Parsippany, N.J.)or a DC stimulation device such as an SpF® XL IIb spinal fusionstimulator (EBI, L.P., Parsippany, N.J.). Pulsed electromagnetic fieldscan be produced using various known methods and apparatuses, such asusing a single coil or a pair of Helmholtz coils. For example, asuitable apparatus includes the EBI Bone Healing System® Model 2001(EBI. L.P., Parsippany, N.J.) and the BTBS stimulation coil. Withrespect to direct current, an electric field may be generated using anyknown device for generating a direct current electric field, such as forexample, the Osteogen™ implantable bone growth stimulator (EBI, L.P.,Parsippany, N.J.). Other suitable devices for generating electromagneticfields may be used.

Electromagnetic stimulation of the cytokine cell suspension can becontinued and/or repeated as desired with respect to contacting theliquid and the solid extraction material. It should be understood,however, that the step of stimulating the liquid with an electromagneticfield includes fields other than, or in addition to, electric orelectromagnetic fields associated with ambient conditions (such theelectromagnetic fields generated by casual exposure to radios,telephones, desktop computers or similar devices).

In some embodiments, both the contacting and stimulating steps as shownin FIG. 1 are performed in less than about 1 hour. The contacting andstimulating steps can also be performed at temperatures ranging fromabout 20° C. to about 37° C. In a preferred embodiment, the temperatureof the cytokine cell suspension is kept at about 37° C. during thecontacting and stimulating steps. One or both of the contacting andstimulating steps are typically performed ex vivo.

Other Methods for Forming Protein Solutions

The present technology provides other methods for forming ProteinSolutions, such as the admixture of proteins and other components andthe isolation and concentration of proteins and components without usingsolid extraction materials. Protein Solutions of the present technologycan be made entirely comprising proteins made by such methods, or byaddition of proteins made by such methods with components or solutionsmade by tissue isolation and processing with solid extraction materials,as described above.

For example, various methods provide acellular or substantiallyacellular Protein Solutions, comprising one or more proteins asdescribed above. Without limiting the scope, mechanism or function ofthe present technology, such acellular anti-inflammatory cytokinecompositions may offer advantages in certain applications, insofar asthey may not create an immunogenic response in subjects to whom they areadministered.

In particular, by way of example, a Protein Solution may compriseinterleukin-1 receptor antagonist (IL-1ra) that is synthetic orrecombinant, or isolated from autologous, allogeneic or xenogeneic bloodor other biologic sources, aside from the methods described above. Forexample, Kineret™ (anakinra) is a recombinant, non-glycosylated form ofIL-1ra, sold by Amgen Manufacturing, Ltd. (Thousand Oaks, Calif.).Various recombinant interleukin-1 inhibitors and methods of treatmentare described in U.S. Pat. No. 6,599,873, Sommer et al., issued Jul. 29,2003; U.S. Pat. No. 5,075,222, Hannum et al., issued Dec. 24, 1991; andU.S. Application Publication No. 2005/0197293, Mellis et al., publishedSep. 8, 2005. In addition, methods for producing IL-1ra from bodyfluids, including the use of autologous fluids, are described in U.S.Pat. No. 6,623,472, Reinecke et al., issued Sep. 23, 2003; U.S. Pat. No.6,713,246, Reinecke et al., issued Mar. 30, 2004; and U.S. Pat. No.6,759,188. Reinecke et al., issued Jul. 6, 2004. When an allogeneicanti-inflammatory cytokine composition is to be generated, multiplesources of IL-1ra from multiple subjects may be pooled together.

More generally, methods for making acellular Protein Solutions cancomprise culturing cells in a cell culture that either naturally produceanti-inflammatory cytokines, such as IL-1ra, or cells that areengineered to produce such cytokines. Non-limiting examples of cellsthat naturally produce anti-inflammatory cytokines include adiposetissue cells, adipocytes, adipose-derived stem cells, stromal cells,bone marrow cells, mesenchymal stem cells, and blood cells.

In various embodiments, cell lines can be engineered to overproduce ananti-inflammatory cytokine. Non-limiting examples of anti-inflammatorycytokines include VEGF, TNF-α, IL-1ra, sTNF-RI, sTNF-RII, PGDF-AB,PDGF-BB, IGF-I, EGF, TGF-β1, sIL-1RII, and HGF. Stable eukaryotic celllines can be generated that overexpress an anti-inflammatory cytokine bytransfecting eukaryotic cells, such as mammalian cells, with recombinantDNA comprising a gene encoding an anti-inflammatory cytokine and aselectable marker. Alternatively, prokaryotes and yeast can beengineered to overexpress an anti-inflammatory cytokine bytransformation with recombinant DNA comprising a gene encoding ananti-inflammatory cytokine and a selectable marker. Transformations andtransfections can be performed with recombinant DNA molecules comprisinga DNA sequencing encoding an anti-inflammatory cytokine, such as IL-1ra,and a selectable marker. Eukaryotic and prokaryotic cells can beengineered to overexpress the anti-inflammatory cytokine constitutivelyor by induction. Methods for expressing anti-inflammatory cytokines,such as IL-1ra, sTNF-RI, and sTNF-RII, and sIL1-RII in eukaryotic andprokaryotic cells are described in U.S. Pat. No. 6,337,072, Ford et al.,issued Jan. 8, 2002; and U.S. Application Publication No. 2001/0053764,Sims et al., published Dec. 20, 2001.

When a IL-1ra gene is transcribed in humans, the mRNA can be splicedinto four variants, resulting in four isoforms of translated IL-1ra. SEQID NOs: 1, 3, 5, and 7 are the cDNAs for IL-1ra isoforms 1-4respectively, and SEQ ID NOs: 2, 4, 6, and 8 are the amino acidsequences of IL-1ra isoforms 1-4 respectively. Collectively, the IL-1raisoforms are referred to as “IL-1ra.” SEQ ID NO: 9 is the cDNA sequencefor sTNF-RI and SEQ ID NO:10 is the amino acid sequence for sTNF-RI. SEQID NO:11 is the cDNA sequence for sTNF-RII and SEQ ID NO:12 is the aminoacid sequence for sTNF-RII. SEQ ID NO:13 is the cDNA sequence forsIL-1RI and SEQ ID NO:14 is the amino acid sequence for sIL-1RI. SEQ IDNOs 15 and 17 are the cDNAs for sIL-1RIIv1 and sIL-1RIIv3 respectively,and SEQ ID NOs:16 and 18 are the amino acid sequences for sIL-1RIIv1 andsIL-1RIIv3 respectively. The cDNA sequence for IL-1RIIv2 is a non-codingsequence; therefore, it is not included.

To express either IL-1ra, sTNF-RI, or sTNF-RII (generically referred toas a “protein of interest”) in a prokaryotic culture, for example in aparticular bacteria, a cDNA sequence (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, or 17) is cloned into an expression vector suitable for thebacteria. The expression vector should comprise a strong promoter, and aselectable marker, such as antibiotic resistance. Non-limiting examplesof antibiotics capable of killing bacteria cells include ampicillin,tetracycline, kanamycin, and chloramphenicol. The expression vectorshould further comprise elements that result in constitutive orinducible expression of the protein of interest. Optionally, a DNAsequence corresponding to a tag functionally coupled to the protein ofinterest that allows for identification and purification of the proteincan be present in the vector adjacent to the gene for the protein ofinterest. For example, an N or C-terminal His tag can be used to detectproteins with anti-His antibodies, and they allow for purification onnickel columns. When the expression vector comprising a gene expressinga protein of interest is prepared, a bacteria cell, for example E. coli,can be transformed with the expression vector. The selectable markerensures that only cells transformed with the vector will survive in LBbroth supplemented with an antibiotic corresponding to the selectablemarker. The bacteria can then be grown in LB broth supplemented with theantibiotic for expression and purification. Expression vectors, methodsfor cloning a protein of interest into an expression vector, methods fortransforming prokaryotic cells, methods for expressing protein fromtransformed prokaryotic cells, and protein purification methods arecommonly known by those with ordinary skill in the art.

To express a protein of interest in a eukaryotic culture, for example inmammalian cells, a cDNA sequence (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,or 17) is cloned into an expression vector suitable for a particularmammalian cell. The expression vector should comprise a strong promoter,and a selectable marker, such as antibiotic resistance. Non-limitingexamples of antibiotics capable of killing mammalian cells includegeneticin and gentamicin. The expression vector should further compriseelements that result in constitutive or inducible expression of theprotein of interest. Optionally, a DNA sequence corresponding to a tagfunctionally coupled to the protein of interest that allows foridentification and purification of the protein can be present in thevector adjacent to the gene for the protein of interest. When theexpression vector comprising a gene expressing a protein of interest isprepared, a mammalian cell, such as a human cell, can be transfectedwith the expression vector. Transfected cells can be grown in a cellculture medium supplemented with an antibiotic corresponding to theselectable marker. The presence of the antibiotic allows for theisolation of stable cell lines. Stable cell lines can then be grown incell culture medium supplemented with antibiotic for expression andpurification. Expression vectors, methods for cloning a protein ofinterest into an expression vector, methods for transfecting eukaryoticcells and developing stable cell lines, methods for expressing proteinfrom transfected eukaryotic cells, and protein purification methods arecommonly known by those with ordinary skill in the art.

Alternatively, eukaryotic cells that have not been genetically alteredby DNA transfection can be cultured. The eukaryotic cells can be primarycultures, i.e. cells grown directly from a eukaryotic donor, such as ahuman, or the eukaryotic cells can be established cell lines. Manyestablished cell lines are available commercially from American TypeCulture Collection, Inc. (Manassas, Va., USA). The cells can be grownwith or an exogenous signal, such as a recombinant protein. Eukaryoticcells are often cultured in culture flasks with cell culture medium. Thecell culture medium can be recovered from the flasks, and centrifuged toremove any non-adherent cells.

A cell culture can be a monolayer culture, a non-adherent culture, or abioreactor. A monolayer culture comprises anchorage-dependent cells thatare cultured on a suitable substrate that allows cell adhesion andspreading, such as cell culture flasks and cell culture dishes. Anon-adherent culture comprises cells that are maintained in asuspension. Suitable cells are either not anchorage-dependent, or theyare anchorage-dependent cells that have been adapted for culture in asuspension. Many cell lines, for example many insect cells, can be grownin either a monolayer or a suspension. A bioreactor is a device that cansupport a biologically active environment in which chemical processesare carried out and/or biochemically active substances are derived.Bioreactors can include suspended or immobilized cells. Monolayercultures, non-adherent cultures, and bioreactors can be maintained bymethods commonly used in the art.

In some embodiments, the cell culture is subjected to an electromagneticfield, so as to stimulate the production of one or more proteins.Stimulating the culture with an electromagnetic field may involvevarious forms of electromagnetic stimulation, such as a pulsedelectromagnetic field or a capacitively coupled electromagnetic field.Methods and conditions for stimulation include those discussed above.

Cell cultures can either release anti-inflammatory cytokines intoculture medium naturally, or the cultures can be induced to release theanti-inflammatory cytokines into the culture medium. The culture mediumcan be isolated by aspiration, centrifugation or filtration to form theacellular anti-inflammatory cytokine composition.

In some embodiments, an anti-inflammatory cytokine is isolated fromurine, for use in producing a Protein Solution of the presenttechnology. Proteins can be isolated from urine by methods among thoseknown in the art. One such method is employed in the ProteoSpin™ UrineProtein Concentration Maxi Kit sold by Norgen Biotek Corp. (Thorold,Ontario, Canada). This kit utilizes an ion exchange resin integratedinto a spin column. Briefly, a urine sample is obtained and its pHadjusted to 3.5. The urine is then transferred to a spin columncontaining the ion exchange resin, which is placed in a collection tube.The column is then centrifuged, wherein the proteins attach to theresin, and the remaining fluids and salts flow into the collection tubeand are discarded. The proteins are then washed by applying suppliedcolumn activation and wash buffer followed by centrifugation. The flowthrough is discarded and the wash procedure is repeated. An elutionbuffer (10 mM sodium phosphate, pH 12.5) is added to the column andneutralizer is added to an elution tube. The spin column containing theelution buffer is placed in the elution tube and centrifuged, wherebythe proteins are eluted and captured in the elution tube containingneutralizer.

Therapeutic Compositions

The present technology also provides compositions comprising a ProteinSolution and a second component comprising active materials,physiological carriers, and combinations thereof. In some embodiments,compositions comprise a safe and effective amount of the ProteinSolution and a safe and effective amount of a second active. A “safe andeffective” amount of a component is an amount that is sufficient to havethe desired therapeutic effect in the human or other mammalian subject,without undue adverse side effects (such as toxicity, irritation, orallergic response), commensurate with a reasonable benefit/risk ratiowhen used in the manner of this technology. The specific safe andeffective amount of the component will, obviously, vary with suchfactors as the particular condition being treated, the physicalcondition of the patient, the nature of concurrent therapy (if any), thespecific components used, the specific route of administration anddosage form, the carrier (if any) employed, and the desired dosageregimen.

Active materials among those useful herein include biologics andpharmaceutical actives. Biologics include blood fractions, such as PRP,blood products, and concentrated bone marrow aspirate (cBMA).

Accordingly, in some embodiments, the present technology providescompositions comprising a safe and effective amount of a ProteinSolution and a safe and effective amount of cBMA. cBMA can includehematopoietic, stem cells, stromal stem cells, mesenchymal stem cells,endothelial progenitor cells, red blood cells, cytokine-producing cells,fibroblasts, reticulocytes, adipose cells, or endothelial cells. Asdescribed above, the Protein Solution may be made using bone marrowaspirate as a cytokine containing tissue. However, a therapeuticcomposition may additionally comprise cBMA with Protein Solution. In oneembodiment, a therapeutic composition comprises a Protein Solution andcBMA in an Protein Solution:cBMA ratio of about 1:1, about 1:2, about1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 orabout 1:10. Alternatively, the Protein Solution:cBMA ratio can be about2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1,about 9:1 or about 10:1. The cBMA and Protein Solution may also beproduced simultaneously. Thus, in reference to FIG. 1 and the processesdescribed above, bone marrow aspirate may be added to the whole bloodobtained in step 115, prior to or during the contacting with a solidextraction material in step 140; such a process involves operation ofboth steps 115 and 130. For example, bone marrow aspirate may be addedto whole blood prior or during isolation of platelet-rich plasma in step120. Such methods include those described in U.S. ApplicationPublication No. 2006/0278588, Woodell-May, published Dec. 14, 2006.

In some embodiments, the cBMA and Protein Solution may be producedsimultaneously. Thus, in reference to FIG. 1 and the processes describedabove, bone marrow aspirate may be added to the whole blood obtained instep 115, prior to or during the contacting with a solid extractionmaterial in step 140; such a process involves operation of both steps115 and 130. For example, bone marrow aspirate may be added to wholeblood prior or during isolation of platelet-rich plasma in step 120.Such methods include those described in U.S. Application Publication No.2006/0278588, Woodell-May, published Dec. 14, 2006.

Pharmaceutical actives among those useful herein include herein includeorganic molecules, proteins, peptides, peptidomimetics, nucleic acids,nucleoproteins, antisense molecules, polysaccharides, glycoproteins,lipoproteins, carbohydrates and polysaccharides, botanical extracts, andsynthetic and biologically engineered analogs thereof, living cells(other than cytokine-producing cells stromal cells) such aschondrocytes, bone marrow cells, viruses and virus particles, naturalextracts, and combinations thereof. Specific non-limiting examples ofbioactive materials include hormones, antibiotics and otheranti-infective agents, hematopoietics, thrombopoietics, antiviralagents, antitumor agents (chemotherapeutic agents), antipyretics,analgesics, anti-inflammatory agents, antiallergy agents, vasodilators,cytokines, growth factors, gene regulators, vitamins, minerals and othernutritionals, nutraceuticals and combinations thereof. In someembodiments, compositions may comprise growth factors in addition tothose present in the Protein Solution, such Platelet-Derived GrowthFactor (PDGF), Transforming Growth Factor Beta (TGF-β), Insulin-LikeGrowth Factor (IGF), Fibroblast Growth Factor (FGF), Epidermal GrowthFactor (EGF), Vascular Endothelial Growth Factor (VEGF), and BoneMorphogenetic Proteins (BMPs).

The compositions may comprise a carrier material, in addition to anyliquid comprising the Protein Solution. It should be understood that invarious embodiments of the present technology, methods of treatmentemploy the Protein Solution as comprised and made above, without furthercarrier, by direct injection or other application to the site oftreatment. However, in other embodiments, an additional carrier materialmay be used for such reasons as for ease of administration, tofacilitate administration using a particular delivery device, enhancingactivity, an increasing the length of time the Protein Solution remainsat the site of administration. Carriers among those useful hereininclude saline, hyaluronic acid, collagen, buffers (such as Hank'sBuffer), cell culture media, blood products (such as PRP and plateletpoor plasma), and mixtures thereof.

Protein Solutions, and compositions comprising Protein Solutions may besterilized prior to administration, by any suitable method. For example,a Protein Solution may be sterilized by including a sterile filter toprocess the product made by the processes described above. In someembodiments, an antibiotic may be included in the solid extractionmaterial during the contacting step described above, or may be added atone or more of the various steps in the methods and treatments describedherein. Alternatively, or in addition, the Protein Solution may beproduced asceptically

Protein Solutions and compositions comprising Protein Solutions may alsobe lyophilized (freeze drying, or cryodesiccation) after production,using methods among those known in the art. Thus, as depicted in FIG. 1,the Protein Solution can be lyophilized after it is isolated from thesolid extraction material. When freeze dried, the anti-inflammatorycytokine composition can be hydrated with a suitable media 170, at atime before administration or at a time of administration. Hydration maybe accomplished by mixing the composition with a solution includingsaline, buffers, blood, blood fractions, bone marrow aspirate,concentrated bone marrow aspirate, and combinations thereof.

The present technology also provides compositions comprising componentsderived from blood or other tissue that are suitable for allogeneicadministration. In particular, such compositions may comprise proteinsand other components isolated from a mammalian subject, or a pluralityof mammalian subjects, other than the subject to whom the composition isto be administered in a method of this technology. In further referenceto FIG. 1, compositions made by contacting a cytokine cell suspensionwith a solid extraction material may be made suitable for allogeneicadministration by freeze drying, as depicted in step 160, afterisolation of the Protein Solution from the solid extraction material. Insome embodiments, the composition can be processed to removecytokine-producing cells present in the Protein Solution compositionafter contacting step 140. Methods for removing cytokine-producing cellsinclude those known in the art, including filtering, clotting, andgravimetric methods. In some embodiments, isolating the blood fractioncomprising plasma and removing cytokine-producing cells are performedessentially simultaneously. Thus, the present technology providesmethods for making a non-immunogenic anti-inflammatory cytokinecomposition, comprising:

-   -   (a) obtaining a cytokine cell suspension from a mammalian donor;    -   (b) contacting the liquid with solid extraction material to        generate a composition rich in interleukin-1 receptor        antagonist;    -   (c) removing cytokine-producing cells from the composition; and    -   (d) freeze drying the composition to produce the non-immunogenic        anti-inflammatory cytokine composition.

In some embodiments, a crypreservative storage solution is added to theProtein Solution, to provide stability for subsequent storage at reducedtemperatures. Suitable storage solutions include those in the art, suchas glycerol and dimethylsulfoxide (DMSO). The composition may be storedat reduced temperatures, such as from about 1° C. to about 6° C. In someembodiments, the composition is stored under liquid nitrogen, at about−80° C. Preferably, the cryopreservative storage solution is removedfrom the Protein Solution prior to administration to a mammaliansubject. Removal of the storage solution may be performed by methodsincluding those known in the art for processing stored blood comprisingcryopreservatives. Washing may be performed using a wash solution, suchas saline. In such embodiments, the blood type of the subject to betreated may be matched to the blood type of the donor from whom thecytokine cell suspension was obtained.

Methods of Treatment

The present technology provides methods for stimulating chondrocyteproduction, including stimulating chondrocyte production at the site ofa collagen defect. Methods include the treatment of collagen defects bystimulating chondrocyte production. Methods comprising administration ofa Protein Solution of the present technology to site of the defect inthe subject. As referred to herein, “treatment” includes one or more ofpreventing enlargement or progression of a defect, reducing the size ofa defect, healing of the defect, and reducing or eliminating painassociated with the defect. Collagen defects may be associated withacute or chronic disorders, and may be associated with an underlyinginjury, trauma, disease, or other physiologic insufficiency of bone,muscle, cartilage, vascular tissue, or other comprising collagen. Invarious embodiments, the pain disorder is associated with aninflammatory disorder, including inflammation mediated by IL1-ra. Suchinflammatory disorders include rheumatoid arthritis, osteoarthritis,osteolytis, tendonitis, synovitis, peripheral vascular disease, andinflammatory respiratory diseases (such as chronic obstructive pulmonarydisease, fibrosis, emphysema, acute respiratory distress syndrome, andpneumonia). Specific pain disorders include pain associated withtraumatic injury, muscle strain, arthritis (rheumatoid arthritis andosteoarthritis), synovitis, sacroiliac joint disorders, back disorders,post-surgical injections, tendon injections, sports medicine procedure(for example, ACL repair, MCL repair, BTB repair, patella repair, orcartilage repair), contusions, muscle strains, post traumaticosteoarthritis.

In some embodiments, methods of the present technology compriseadministration of a Protein Solution to the site of a collagen tissuedefect. As referred to herein, such “tissue defects” include anycondition involving tissue which is inadequate for physiological orcosmetic purposes. Examples of such defects include those that arecongenital, those that result from or are symptomatic of disease,disorder, or trauma, and those that are consequent to surgical or othermedical procedures. Embodiments include treatment for vascular, bone,skin, nerve, and organ tissue defects. Examples of such defects includethose resulting from arthritis (osteoarthritis and rheumatoidarthritis), spinal fixation procedures, hip and other joint replacementprocedures, chronic wounds, fractures, sclerosis of tissues and muscles,and spinal cord or other nerve injury. In various embodiments, thecompositions and methods of this invention may be used in methodsassociated with the repair bone or cartilage defects. For example, thepresent technology provides methods for stimulating chondrocyteproduction at the site of a joint afflicted with osteoarthritis,comprising administering a Protein Solution at or near the joint.

In various embodiments, methods are for the treatment of cartilagedefects in a human. In other embodiments, treatment is of a defect in anon-human mammal, such as a companion, working, or sports animal. Forexample, such methods of this technology may be used for treatment of acollagen defect associated with a joint injury in a horse.

In various embodiments, methods of the present technology comprise apoint-of-care method for making a Protein Solution. As referred toherein, a “point-of-care method” wherein the processes of the presenttechnology are performed at a time proximate to the administration ofthe Protein Solution to the subject being treated. Such methods may beperformed at a location proximate, such as in the same room (forexample, bed side) or otherwise immediately adjacent, to the mammaliansubject to be treated with the Protein Solution. In various embodiments,a “proximate time” may be, for example, within 12 hours, within 8 hours,within 2 hours, within 1 hour or within 30 minutes of administration ofthe Protein Solution to the subject.

In some embodiments, the Protein Solution is administered with aconcomitant therapy. Such therapies include, for example, theadministration of pharmaceutical actives or biologics, as describedabove. In some embodiments, concomitant therapies are administeredconcurrently with a Protein Solution. For example, methods may compriseadministration of a Protein Solution with a safe and effective amount ofan active selected from the group consisting of analgesics andglucocorticosteroids.

In some embodiments, methods comprise administration of a ProteinSolution with concentrated bone marrow aspirate, as described above. Forexample, cBMA and a Protein Solution may be administered concomitantly.

Methods of the present technology generally comprise administration of aProtein Solution to the site of collagen defect in a mammalian subject.Administration of the Protein Solution can be performed with anysuitable device, including such devices known in the art for topicaldelivery of compositions to the muscle, joint, vascular, or othertissue. For example, topical delivery for treatment of a cartilagedefect in a joint may comprise injection of a Protein Solution at ornear the joint.

Embodiments of the present technology are further illustrated throughthe following non-limiting examples.

Example 1 Preparing and Characterizing a Protein Solution

A Protein Solution rich in interleukin-I receptor antagonist is preparedfrom seven consented human providers. Blood (55 mL) is drawn into a 60cc syringe with 5 mL of anticoagulant citrate dextrose solution A(ACD-A, Citra Labs. Braintree, MA). Platelet-rich plasma (PRP) iscreated using the GPS® III platelet concentration system (800-1 003A,Biomet Biologics, Warsaw, Ind.) according to the instructions for use.The solution is generated by adding 6 mL of PRP to a modified Plasmaxdevice containing 1 gram of polyacrylamide beads (Biomet Biologics,Warsaw, Ind.). The IL-1ra solution is removed from the Plasmax devicesand frozen at minus 50° C. for the assay. Cytokine content is assayed ona 16-plex ELISA (Searchlight Protein Array, Aushon Biosystems,Billerica, Mass.). The analytes included IL-4, IL-10, IL-11, IL-13,IL-1ra, IFN-γ, sTNF-RI, sTNF-RII, IL-1α, IL-1β, TNF-α, IL-17, IL-18,bFGF, TBF-β1, and TBF-β2.

The solution contains both anabolic (bFGF, TGF-β1, TGF-β2 (see Table 3))and anti-inflammatory (IL-1ra, sTNF-RI, sTNF-RII, IL-4, IL-10, IL-11,IL-13, IFN-γ, (see Table 4)) cytokines without expressing large doses ofcatabolic cytokines (IL-1α, IL-1β, TNF-α, IL-17, IL-18 (see Table 5)).The anti-inflammatory cytokines IL-1ra and sTNF-R are all detected inng/mL quantities, while all of the catabolic analytes were in pg/mLquantities. However, donor-to-donor variability is detected.Correlations between the catabolic cytokines IL-1 and TNF-α andanti-inflammatory analytes IL-1ra and sTNF-R are compared, but no largecorrelations detected (Table 6). On average, there is about 13,260 timesmore IL-1ra than IL-1α and about 7,561 times more than IL-1β.

TABLE 3 Anabolic cytokines in the solution. Donor bFGF TGF-β1 TGF-β2 118.5 1,458,008 153,833 2 10.7 1,137,404 119,545 3 11.9 585,298 70,544 44.9 1,342,442 162,707 5 20.0 1,579,361 204,670 6 7.7 1,393,746 170,345 713.9 1,474,155 174,502 Average 12.5 1,281,488 150,878 ±SD ±5.5 ±336,345±43,617

TABLE 4 Anti-inflammatory cytokines in the solution. TNF- Donor IFN-γIL-4 IL-10 IL-13 IL-1ra TNF-RI RII IL-11 1 <0.4 2.1 0.5 3.5 9,660 2,7282,249 <2.0 2 <0.4 1.3 0.3 2.8 17,477 5,120 2,900 <2.0 3 <0.4 <0.8 0.30.1 23,126 6,247 2,446 <2.0 4 40.4 59.9 8.9 19.9 10,458 4,374 2,612 <2.05 30.2 33.9 23.3 15.8 13,462 2,763 1,394 <2.0 6 2.6 23.3 1.4 25.6 8,8132,992 2,716 <2.0 7 0.7 1.2 0.6 1.8 11,277 3,330 1,915 <2.0 Average 10.717.5 5.0 9.9 13,468 3,936 2,319 <2.0 ±SD ±17.0 ±22.9 ±8.7 ±10.3 ±5,154±1,356 ±520 ±0

TABLE 5 Catabolic cytokines in the solution. Donor IL-17 TNF-α IL-1αIL-1β IL-18 1 3.1 16.0 <0.8 1.5 239 2 1.2 <2.3 2.5 3.3 559 3 0.7 <2.31.8 2.3 511 4 28.9 195 0.8 1.3 329 5 33.8 661 0.8 2.0 450 6 22.0 1.050.3 1.7 333 7 6.7 <2.3 1.9 1.0 787 Average 13.8 141 1.3 1.9 458 ±SD±14.1 ±241 ±0.8 ±0.8 ±183

TABLE 6 Correlation analysis. Analytes compared R² Ratio IL-1ra andIL-1α 0.46 13,260X   IL-1ra and IL-1β 0.45 7,561X   TNF-RI and TNF- α0.17 945X TNF-RII and TNF- α 0.47 477X

Example 2 Generation of IL-1Ra from Platelet-Rich Plasma

An IL-1ra-rich solution is created as follows. Whole blood (70 mL)anticoagulated (10%) with ACD-A (Braintree, Mass., USA) is drawn from 5healthy volunteers. A portion (10 mL) is reserved for a whole bloodmeasurement. Platelet-rich plasma (PRP) (6 mL) is produced using theGPS® II System (Biomet Biologics, LLC, Warsaw, Ind., USA). Completeblood counts are collected for the whole blood and PRP samples followinga validated procedure, as described in Woodell-May JE, Ridderman DN,Swift MJ, Higgins J. “Producing Accurate Platelet Counts for PlateletRich Plasma: Validation of a Hematology Analyzer and PreparationTechniques for Counting” J. Craniofac. Surg. (2005) September16(5):749-56.

Following the PRP production, 5 mL of the PRP is added to a modifiedplasma concentration device (Plasmax™, Biomet Biologics LLC, Warsaw,Ind., USA) and incubated with polyacrylamide desiccating beads in thedevice for 24 hours at room temperature. Following the contact withpolyacrylamide beads the electromagnetic field, the plasma concentrationdevice is centrifuged to separate the serum fraction.

To analyze baseline IL-1ra levels at time zero, the whole blood and PRPsamples are activated with 50 μL of thrombin and 10% CaCl2 (1,000units/mL). A blood clot is formed and incubated for 30 minutes at roomtemperature. Following incubation, the clot is centrifuged for 5 minutesat 3,000 rpm. Serum is collected from the clots and retained for ELISAanalysis. The serum fraction from the plasma concentrator does notrequire activation by thrombin, and is tested directly. All samples areanalyzed for IL-1ra using an ELISA kit (IL-1ra Quantikine™ Kit, R&DSystems, Minneapolis, Minn., USA).

The PRP samples result in about an eight-fold increase in platelets,about five-fold increase in total cytokine-producing cells (WBCs), aboutnine-fold increase in the monocyte fraction of the WBCs, and about athree-fold increase in the PMN fraction of the WBCs. The IL-1raproduction in the whole blood and PRP samples is correlated most closelyto the WBC concentration. The five-fold increase in the PRP is likelydue to the increase in WBCs, and both the whole blood and PRP IL-1ravalues can be considered baseline IL-1ra content. This is in contrast tothe 195-fold increase in IL-1ra following incubation in the plasmaconcentrator. This plasma concentration device typically results in a3-fold increase in plasma protein concentration due to a volumereduction caused by the desiccation process. This 3-fold decrease involume does not account for the levels of increase seen in the amount ofIL-1ra. Therefore, this level of increase indicates stimulation of WBCsto produce IL-1ra during the contact with the solid extraction material(e.g., polyacrylamide beads) and electromagnetic field stimulation.

Correlation analysis demonstrates that IL-1ra production is more closelycorrelated with the increase in WBCs than the platelet content. TheIL-1ra levels do not correlate as closely with the WBC population in thePRP. This is not surprising since the WBC are not activated, and theserum is collected by thrombin activation of the plasma. However, it ispossible that the WBC, once activated in the plasma concentrationdevice, participate in the significant production of IL-1ra seen.

Example 3 Production of Protein Solution from PRP

Anticoagulated blood (120 cc) is collected from 5 human donors.Platelet-rich plasma (PRP) is prepared using GPS® III disposables(Biomet Biologics LLC, Warsaw, Ind., USA). PRP is loaded into modifiedplasma concentration devices (Plasmax™, Biomet Biologics LLC, Warsaw,Ind., USA) and processed. The output is divided into 4 groups: IL-1ra inconcentrated plasma with and without thrombin activation (1000 U/mL in1M CaCl2), or cell-free IL-1ra with and without thrombin activation.IL-1ra is measured using ELISA (R&D Systems) over time.

The PRP contacts polyacrylamide beads in the Plasmax™ device whileelectromagnetic field stimulation is provided using a capacitivelycoupled electromagnetic field.

Unclotted PRP produces an average of about 50 ng over 24 hrs. Thecell-free samples produce about 34 ng without changing over 24 hrs. Onceclotted, the elution of IL-1ra is slowed, with only about 30% beingeluted after 10 hours. Release in the cell-free samples is also delayed,but eluted 100% of available IL-1ra after 10 hours.

Example 4 Generation of Protein Solution and Characterization ofCytokine Levels in Healthy Subjects and Osteoarthritis Subjects

An Autologous Protein Solution (APS) from healthy patients are preparedas follows for the measurement of growth factors. 72 ml ofanticoagulated whole blood are drawn by venipuncture from each of sixdonors. 3 ml of each donor's anticoagulated whole blood are aliquotedinto microcentrifuge tubes and frozen at −50° C. 60 ml of theanticoagulated whole blood is loaded into GPS® III disposable devices(Biomet Biologics LLC, Warsaw, Ind., USA), which is processed accordingto the manufacturer's instructions to produce PRP. The PRP is removedfrom the GPS® III devices and added to Plasmax™ devices (BiometBiologics LLC, Warsaw, Ind. USA), which is processed according to themanufacturer's instructions to produce APS. APS is extracted from eachdevice, aliquoted into microcentrifuge tubes, and frozen at −50° C. Eachsample, whole blood and PRP, is subjected to three freeze-thaw cycles.Quantikine Human Immunoassays (R&D Systems, Inc., Minneapolis, Minn.)for VEGF, PDGF-BB, PDGF-AB, EGF, TGF-β1, TGF-β2, and IGF-1 are run induplicate according to the manufacturer's instructions for each APS andwhole blood sample.

APS from healthy patients is prepared as above for the measurement ofanti-inflammatory cytokines. Quantikine Human Immunoassays (R&D Systems,Inc., Minneapolis, Minn.) for IL-1ra, IL-1β, IL-8, sTNF-RI, TNF-α, IL-6,sTNF-RII, IL-10, IL-13, and IL-4 are run in duplicate according to themanufacturer's instructions for each APS and whole blood sample.Immunoassays are also performed to detect hepatocyte growth factor (HGF)and soluble IL-1RII.

APS from 105 osteoarthritis patients is prepared as above for themeasurement of growth factors anti-inflammatory cytokines. The APS isstored at −50° C. or in dry ice.

Cytokine concentrations are compared between healthy donors and OApatients in baseline blood and APS. IL-1β is concentrated at a higherlevel in OA patients, but the fold increase is still much lower thanthat of IL-1ra. Other cytokines and growth factors that are concentratedat least to the level of that observed in healthy donors includesTNF-RI, IGF-I, IL-8, VEGF, and IL-6. The soluble cytokines sTNF-RII andsIL-1RII are concentrated to a level not quite as high but very similarto the healthy concentration level. The results are displayed in Table7.

TABLE 7 Concentration of growth factors and anti-inflammatory cytokinesfrom APS derived from healthy patients and patients with osteoarthritis(in pg/ml). Fold Baseline APS Increase Cytokine Average StDev AverageStDev Average VEGF Healthy 276 109 742 494 2.7 OA 484 201 1710 1025 3.8IL-1β Healthy 3.4 2 3.8 0.8 1.1 OA 3.3 1.1 8.9 7.3 2.8 IL-8 Healthy 7416 315 198 4.3 OA 73.5 29.6 287.9 192.7 4.2 IL-6 Healthy 3.1 0.4 3.4 0.71.1 OA 1.8 1.3 3 3.5 1.6 TNF-α Healthy ND ND 3.4 0.7 ND OA 2.4 2 4.3 35.3 IL-1ra Healthy 8092 2536 30853 16737 3.8 OA 7576 2469 41896 196695.9 sTNF- Healthy 2485 338 9491 1387 3.8 RII OA 1491 492 5060 1946 3.5PDGF- Healthy 13400 3400 91700 24100 6.8 AB OA 16799 5731 37889 249222.5 PDGF- Healthy 4702 1027 23810 6126 5.1 BB OA 5306 2422 11936 86552.5 IGF-I Healthy 114000 30000 155000 34000 1.4 OA 79072 22137 11806042827 1.5 EGF Healthy 240 71 1227 300 5.1 OA 374 199 707 489 2.2 sTNF-Healthy 629 76 2408 338 3.8 RI OA 808 275 3011 964 3.9 TGF-β1 Healthy25717 11131 181245 56420 7.1 OA 56594 56940 153567 145973 4.2 sIL-Healthy 11,786 ND 26,000 ND 2.2 1RII OA ND ND ND ND ND HGF Healthy 782ND 3244 ND 4.1 OA ND ND ND ND ND

Example 5 Generation of a Protein Solution from Adipose Tissue

Adipose stromal cells are prepared as follows. Adipose tissue is mincedinto small pieces (about 1 cm3) and digested in 2 mg/mL type Icollagenase (Worthington Biochemical Corp., Lakewood, N.J.) underintermittent mechanical agitation in a water bath at 37° C. for 180minutes. Digestion can be neutralized by the addition of medium or ablood-derived solution. The cell suspension is centrifuged (300×g for 7minutes at 25° C.) followed by removal of the supernatant from the cellpellet. The pellet is then re-suspended in a compatible solution toprovide a liquid volume comprising adipose stromal cells.

Alternatively, the pellet is suspended with whole blood obtained fromthe subject, and added to a GPS™ Platelet Concentrate System, fromBiomet Biologics, Inc. (Warsaw, Ind.). Following centrifugation, theplatelet-rich plasma layer, which also contains the adipose stromalcells, is extracted from the system.

The adipose stromal cells, optionally including platelet-rich plasma,are then combined with polyacrylamide beads and subjected to a pulsedelectromagnetic field by using a pair of Helmholtz coils to stimulateproduction of IL-1 ra. The adipose stromal cells and polyacrylamidebeads are separated from the liquid solution to obtain a solution richin IL-1ra.

Example 6 Generation of Protein Solution from Lipoaspirate

A therapeutic composition of IL-1ra is generated from stromal cellsisolated from adipose tissue. Isolation of human stromal cells isperformed by obtaining human subcutaneous adipose tissue fromlipoaspiration/liposuction procedures and digesting the tissue incollagenase type I solution (Worthington Biochemical Corp., Lakewood,N.J.) under gentle agitation for 1 hour at 37° C. The dissociated cellsare filtered with 500 μm and 250 μm Nitex filters. The fraction iscentrifuged at 300×g for 5 minutes. The supernatant is discarded and thecell pellet is re-suspended in a compatible liquid solution, such as ablood-derived solution.

Non-Limiting Discussion of Terminology

The headings (such as “Introduction” and “Summary”) and sub-headingsused herein are intended only for general organization of topics withinthe present disclosure, and are not intended to limit the disclosure ofthe technology or any aspect thereof. In particular, subject matterdisclosed in the “Introduction” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

The disclosure of all patents and patent applications cited in thisdisclosure are incorporated by reference herein.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Equivalent changes,modifications and variations of specific embodiments, materials,compositions and methods may be made within the scope of the presenttechnology, with substantially similar results. Moreover, recitation ofmultiple embodiments having stated features is not intended to excludeother embodiments having additional features, or other embodimentsincorporating different combinations of the stated features. Specificexamples are provided for illustrative purposes of how to make and usethe compositions and methods of this technology and, unless explicitlystated otherwise, are not intended to be a representation that givenembodiments of this technology have, or have not, been made or tested.

As used herein, the words “prefer” or “preferable” refer to embodimentsof the technology that afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the technology.

As used herein, the word “include,” and its variants, is intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices, and methods of this technology. Similarly, theterms “can” and “may” and their variants are intended to benon-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present technology that do not contain those elements orfeatures.

Although the open-ended term “comprising,” as a synonym ofnon-restrictive terms such as including, containing, or having, is usedherein to describe and claim embodiments of the present technology,embodiments may alternatively be described using more limiting termssuch as “consisting of” or “consisting essentially of.” Thus, for anygiven embodiment reciting materials, components or process steps, thepresent technology also specifically includes embodiments consisting of,or consisting essentially of, such materials, components or processesexcluding additional materials, components or processes (for consistingof) and excluding additional materials, components or processesaffecting the significant properties of the embodiment (for consistingessentially of), even though such additional materials, components orprocesses are not explicitly recited in this application. For example,recitation of a composition or process reciting elements A, B and Cspecifically envisions embodiments consisting of, and consistingessentially of, A, B and C, excluding an element D that may be recitedin the art, even though element D is not explicitly described as beingexcluded herein. Further, as used herein the term “consistingessentially of” recited materials or components envisions embodiments“consisting of” the recited materials or components.

A” and “an” as used herein indicate “at least one” of the item ispresent; a plurality of such items may be present, when possible.“About” when applied to values indicates that the calculation or themeasurement allows some slight imprecision in the value (with someapproach to exactness in the value; approximately or reasonably close tothe value; nearly). If, for some reason, the imprecision provided by“about” is not otherwise understood in the art with this ordinarymeaning, then “about” as used herein indicates at least variations thatmay arise from ordinary methods of measuring or using such parameters.

As referred to herein, ranges are, unless specified otherwise, inclusiveof endpoints and include disclosure of all distinct values and furtherdivided ranges within the entire range. Thus, for example, a range of“from A to B” or “from about A to about B” is inclusive of A and of B.Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatParameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if Parameter X is exemplified herein to have values in the range of1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may haveother ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3,3-10, and 3-9.

1. A method for stimulating chondrocyte production in a mammaliansubject, comprising administering at or near a site of a collagen defectin the mammal a composition comprising at least two proteins selectedfrom soluble interleukin-1 receptor II (sIL-1RII), tumor necrosis factorreceptor II (TNF-RII), epidermal growth factor (EGF), hepatocyte growthfactor (HGF), and platelet-derived growth factor-BB (PDGF-BB), whereinthe concentration of each of the selected at least two proteins in thecomposition is greater than the concentration of the respective ones ofthe selected at least two proteins in normal blood, and wherein thecomposition stimulates chondrocyte production at or near the site of thecollagen defect so as to prevent enlargement or progression of thecollagen defect, reduce the size of the collagen defect, heal thecollagen defect, or a combination thereof.
 2. The method according toclaim 1, wherein one or more of the selected at least two proteins isautologous to the mammal.
 3. The method according to claim 1, whereinthe composition further comprises at least about 10,000 pg/mlinterleukin 1 receptor antagonist (IL-1ra); and a protein selected fromtumor necrosis factor receptor 1 (TNF-RI), insulin-like growth factor-1(IGF-1), platelet-derived growth factor-AB (PDGF-AB), vascularendothelial growth factor (VEGF), transforming growth factor-β1(TGF-β1), and a mixture thereof, wherein the selected protein has aconcentration higher than the selected protein's concentration in normalblood.
 4. A method for stimulating chondrocyte production in a mammaliansubject, comprising administering at or near a site of a collagen defectin the mammal a composition comprising: interleukin-1 receptorantagonist (IL-1ra) at a concentration at least 3 times greater than theconcentration of IL-1ra in normal blood; soluble tumor necrosis factorreceptor-I (sTNF-RI), at a concentration at least 2 times greater thanthe concentration of IL-1 ra in normal blood; cytokine-producing cellsat a concentration at least 2 times greater than the concentration ofcytokine-producing cells in normal blood; platelets, at a concentrationat least 2 times greater than the concentration of platelets in normalblood; and a protein selected from epidermal growth factor (EGF),hepatocyte growth factor (HGF), platelet-derived growth factor-BB(PDGF-BB), soluble interleukin-1 receptor II (sIL-1RII), tumor necrosisfactor receptor II (TNF-RII) and a combination thereof, wherein theconcentration of the selected protein in the composition is greater thanthe concentration of the selected protein in normal blood, and whereinthe composition stimulates chondrocyte production at or near the site ofthe collagen defect so as to prevent enlargement or progression of thecollagen defect, reduce the size of the collagen defect, heal thecollagen defect, or a combination thereof.
 5. The method according toclaim 4, wherein the concentration of IL-1ra is from about 10,000 pg/mlto about 50,000 pg/ml.
 6. (canceled)
 7. The method according to claim 4,wherein the concentration of sTNF-RI is about 2000 pg/ml or greater. 8.(canceled)
 9. The method according to claim 4, wherein the compositionfurther comprises concentrated bone marrow aspirate.
 10. A method forstimulating chondrocyte production in a mammalian subject, comprising:obtaining a cytokine cell suspension from the subject; fractionating theliquid to produce an autologous protein solution (APS) comprisinginterleukin-1 receptor antagonist (IL-1ra) and a protein selected fromepidermal growth factor (EGF), hepatocyte growth factor (HGF),platelet-derived growth factor-BB (PDGF-BB), soluble interleukin-1receptor II (sIL-1RII), tumor necrosis factor receptor II (TNF-RII), anda combination thereof, wherein the concentration of the selected proteinin the composition is greater than the concentration of the selectedprotein in the subject's whole blood; and administering the autologousprotein solution at or near a site of a collagen defect in the subject,wherein the composition stimulates chondrocyte production at or near thesite of the collagen defect so as to prevent enlargement or progressionof the collagen defect, reduce the size of the collagen defect, heal thecollagen defect, or a combination thereof.
 11. The method according toclaim 10, wherein the cytokine cell suspension comprises a liquidselected from whole blood, bone marrow aspirate, adipose tissue, urine,a fraction thereof, and a mixture thereof.
 12. The method according toclaim 11, wherein the liquid comprises a platelet rich plasma fractionthat includes cytokine-producing cells.
 13. The method according toclaim 10, wherein fractionating comprises placing blood in a separatoroperable to separate the blood into two or more fractions; and operatingthe separator to create a platelet-rich plasma fraction including whiteblood cells.
 14. The method according to claim 13, wherein fractionatingcomprises contacting platelet-rich plasma with polyacrylamide beads, andseparating the polyacrylamide beads from the platelet-rich plasmafraction to form the APS.
 15. The method according to claim 10, whereinthe cytokine cell suspension comprises whole blood, bone marrowaspirate, or both, and fractionating comprises: loading the cytokinecell suspension into a separator comprising a buoy, wherein the buoy hasa density such that the buoy reaches an equilibrium position between afirst fraction and a second fraction of the cytokine cell suspension;operating the separator so that the buoy defines an interface betweenthe first fraction and the second fraction, the second fraction having aconcentration of cytokine-producing cells greater than the concentrationof cytokine-producing cells in the first fraction; collecting the secondfraction; contacting the second fraction with a solid extractionmaterial the to form the APS; and separating the solid extractionmaterial from the APS, wherein the APS has an IL-1ra concentrationgreater than the IL-1ra concentration in normal whole blood.
 16. Themethod according to claim 15, wherein the solid extraction material isselected from corundum, quartz, titanium, dextran, agarose,polyacrylamide, polystyrene, polyethylene, polyvinyl chloride,polypropylene, and a combination thereof.
 17. (canceled)
 18. The methodaccording to claim 15, wherein the solid extraction material comprises aform selected from a bead, fiber, powder, porous material, and acombination thereof.
 19. The method according to claim 10, furthercomprising: obtaining bone marrow aspirate from the subject;concentrating the bone marrow aspirate to produce a concentrated bonemarrow aspirate (cBMA); administering the cBMA to the subject at or nearthe site of the collagen defect.
 20. The method according to claim 19,wherein administering of the cBMA is performed concurrently withadministering of the APS.
 21. The method according to claim 20, whereinthe APS and the cBMA are in an APS:cBMA ratio of from about 10:1 toabout 1:10.
 22. The method according to claim 19, wherein the bonemarrow aspirate is mixed with the cytokine cell suspension during orprior to fractionating the cytokine cell suspension.
 23. The methodaccording to claim 9 wherein fractionating comprises placing thecytokine cell suspension and the bone marrow aspirate in a separatoroperable to separate the cytokine cell suspension and bone marrowaspirate into two or more fractions; and operating the separator tocreate a platelet-rich plasma fraction comprising white blood cells andthe cBMA. 24.-27. (canceled)
 28. The method according to claim 10,further comprising topically administering at or near the site of thecollagen defect a composition comprising hyaluronic acid, collagen, or acombination thereof.